The polyspecific immunoglobulin response to HSV-1 viral proteins: determination of immunogenic proteins and relative antibody titres to individual polypeptides by immunoblotting

Abstract

Summary.We developed an immunoblotting procedure to characterise the polyspecific immunoglobulin response to the proteins of herpes simplex virus (HSV)-1 . We found that 8-20% gradient polyacrylamide gels provided no advantage over fixed 8.5% gels for preparing Western blots for use in immunoblotting. The amount of protein loaded on the gels markedly influenced which proteins were detected by immune serum. The presence of Triton-X-100 0.5% in washes and buffers improved band clarity on immunoblots. In optimal conditions, immune mouse serum reacted with up to 33 HSV-1 lysate proteins. Six bands or regions appeared to be of major immunogenic reactivity, including the (122-130) x 103-mol. wt region, a 75 x 103-mol. wt protein, the gD region of approximately (56-64) x 103-mol. wt and two non-glycosylated bands at mol. wts( lo3) 42 and 44. Minor proteins, more weakly reactive, were detected at 27 other areas. The relative antibody titres in immune mouse serum to the different major regions showed antibody titre to gD > gB/gC > (42/44) x lo3 >> P75 >> VP154.Most human sera reacted with all of the major and many of the minor immunogenic proteins but individual sera varied markedly in the proteins recognised. We conclude that immunoblotting is valuable for evaluating immunoglobulin responses to major and minor immunogenic proteins of HSV-1.

Murine IgG Subclass Responses to Herpes Simplex Virus Type 1 and Polypeptides

“…HSV-l-infected cell lysate proteins were loaded at 11 gg/4 mm lane width in gels 0.75 mm thick and 14 cm long. Proteins were identified on the basis of their SDS-PAGE migration relative to known Mr markers, and for glycoproteins, by comparison with lanes containing [14C]glucosamine-labelled infected cell lysate proteins (McKendall et al, 1988a). Proteins were transferred to nitrocellulose membranes as described by Towbin et al (1979), at 25 V, 0.2 A overnight.…”

“…Proteins were transferred to nitrocellulose membranes as described by Towbin et al (1979), at 25 V, 0.2 A overnight. Optimal time and current were determined as previously reported (McKendall et al, 1988a).…”

Antibody response to herpes simplex virus glycoproteins gB and gD

“…HSV-1 glycoproteins gB and gD are cross-reactive with HSV-2 gB and gD and induce a strong humoral response following both HSV-1 and HSV-2 infections [Bernstein et al, 1984;Eberle and Mou, 1983;Ashley et al, 1985, Kahlon et al, 1986McKendall et al., Fig. 1.…”

Antibody activity to herpes simplex virus in mouse Ig classes and IgG subclasses

“…In general numerous proteins are highly immunogenic including the major glycoproteins [5,10,16,20,22]. In our studies, using high resolution gels and Western blots carefully controlled for optimum load of viral proteins, we identified 33 polypeptides which were reactive with antibodies in immune mouse serum [19]. The most immunogenic proteins were 154KDa (the major capsid protein), gC, gB, pgB, 75KDa, gD and nucleocapsid complex proteins of 42KDa and 44KDa dalton molecular weights.…”

“…Production of HSV-1 polypeptide containing cell lysates, separation of HSV-1 polypeptides by SDS-polyacrylamide gel electrophoresis, and electrophoretic transfer of proteins to nitrocellulose were performed, as previously described [19] using modifications of the methods of Laemmli [14] and Towbin et al [28]. The Western blots were cut into strips, placed into troughs and non-specific binding was blocked with blocking buffer [PBS, pH 7.22 with 3% (w/v) BSA, 0.2% (w/v) sodium azide, 0.5% (v/v) Triton-X-100].…”