Figure 2
Immunological and structural analyses of the L1 deletion and its derivatives. A) Schematic diagram of the full-length 16 L1 protein (VLP) and three L1 derivatives: a deletion lacking residues 404–436 (C-1), and those bearing RSV F aa 255–278 (3-1) and 423–436 (423-3) within the C-1 deletion. B and C) ELISA analysis of L1 derivatives: mouse polyclonal anti-HPV 16 VLP (Panel B; starting at 1:10,000) and mouse mAb V5 (Panel C; starting at 1:100,000) were used to detect L1 VLPs and the capsomere derivatives. Horizontal axis represents serial two-fold dilutions of antibodies and vertical axis represents OD405 nm of the resulting reactions. D) Immunoblots of L1 VLP and capsomere derivatives. Except for the 423-3 lane in the right panel, ~0.5 μg/lane of L1 proteins were resolved on 10%/5% (left and right panels) or 12%/6% (middle panel) SDS-PAGE gels, transferred onto nitrocellulose, and detected with anti-16L1 mAb (CAMVIR-1;1:60,000 dilution), anti-RSV F neutralizing mAb (L4; 1:5,000 dilution), or anti-RSV F polyclonal rabbit serum (1:1,000 dilution) followed by either goat anti-mouse IgG-HRP or anti-rabbit IgG-HRP (at 1:20,000 dilution) and chemiluminescence. Molecular weight standards are shown to the left of each marker ladder. The two anti-RSV F antibodies used in this study recognize both the 50 kD and 20 kD subunits of F protein. Compared to other lanes, 3–4 fold more total protein of the 423-3 derivative was loaded; similar over-loading of C-1 preparations did not lead to increased non-specific recognition of capsomeres by the polyclonal anti-RSV serum (data not shown). The multiple L1 bands around 55 kD likely indicate minor differences in post-translational modifications of the L1 derivatives.