Synthesis of a Fluorescent Ganglioside G_M1 Derivative and Screening of a Synthetic Peptide Library for G_M1 Binding Sequence Motifs

A ganglioside GM1 probe bearing a dark-red fluorescent dye at the sphingosine moiety of the molecule was prepared by a convenient one-pot synthesis. The labeled GM1 permitted the detection of the natural ganglioside GM1 ligand Escherichia coli heat-labile enterotoxin subunit B (EtxB) in picomole quantities on a solid support. When an epitope mapping of several ganglioside binding proteins and protein fragments was performed by screening a cellulose membrane-bound synthetic library of 64 16mer peptides with the new probe, several peptides displaying ganglioside GM1 affinity could be identified. We consider the labeled glycolipid described herein a versatile tool for manifold biochemical investigations

Phage-display derived single-chain fragment variable (scFv) antibodies recognizing conformational epitopes of Escherichia coli heat-labile enterotoxin B-subunit

Previously we have described studies on in vitro pentamer assembly of Escherichia coli (E. coli) derived heat-labile enterotoxin B subunit (EtxB) using conventional monoclonal antibodies (Amin et al., JBC 1995: 270, 20143-50 and Chung et al., JBC 2006: 281, 39465-70). To extend these studies further we have used phage-display to select single-chain Fragment variable (scFv) antibodies against different forms of the B-subunit. Two clones exhibiting strong and differential binding were chosen for detailed characterization. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions and a nonsense mutation present in one of these (scFv-B1.3.9) was corrected. Binding analysis showed that scFv-B1.3.9 bound in ELISA to both heat-denatured monomeric B-subunits (EtxB1) and also displayed cross-reactivity towards pentameric EtxB (EtxB5), although there was no reactivity towards monoganglioside (GM1) captured EtxB5. Another antibody (scFv-B5.2.14) had a different reactivity profile and, in ELISA, bound only to EtxB5 but not to EtxB1 or to EtxB5 captured via GM1. Surprisingly, in competition experiments, the assembled pentameric B-subunit inhibited binding of scFv-B5.2.14 to immobilized EtxB5 only weakly, whereas reduced, but not oxidized, monomeric EtxB1 was an efficient competitor. These results clearly demonstrate that B1.3.9 and B5.2.14 have different specificities for cryptic epitopes not accessible in the fully assembled GM1 bound pentameric form of EtxB. Taken together our results show that we were able to successfully isolate and characterize recombinant scFvs that differentially recognize diverse denatured forms or assembly intermediates of the heat-labile enterotoxin B subunit of E. coli

The ability to detect very low FFU of live RV.

<p>Monolayers of MA104 cells were incubated for 24h with 0.2 to 2000 FFU live RV. Uninfected MA104 cells were used as a negative control. Culture supernatant was used to infect new MA104 monolayers, and previously infected monolayers were visualised by FFA. DAPI (blue) indicates cell nuclei and FITC (green) indicates RV infection. Stained monolayers were visualised using Nikon TiE inverted fluorescent microscope. Scale bar = 100μm. Images representative of 3 replicates per MOI for each passage.</p

Inactivation curves of RV and SFV following exposure to γ-irradiation.

<p>SFV and RV samples were exposed to increased doses of γ-irradiation on dry-ice, and the reduction in virus titre was determined by (A) plaque forming assay for SFV, or (B) fluorescent focus assay for RV. All samples tested in triplicate and data presented as mean ± SEM.</p

Neutralising antibody responses induced by γ-RV.

<p>Mice were primed with live RV or γ-RV twice, 2 weeks apart. Serum samples harvested on Day 21 post-2^nd priming, and neutralising ability of immune serum determined by <i>in vitro</i> neutralisation assay. (A) FFU/well determined following incubation of MA104 cells with sera treated-RV at MOI 0.005. RV treated with serial dilutions of HI control or immune sera. PBS-treated RV used to indicate the baseline level of infection. Data presented as mean ± SEM (n = 2), and analysed by One-Way ANOVA (****, p < 0.0001 compared to naïve control sera for each dilution. #, p < 0.05, when directly comparing immune sera groups). (B) Representative fluorescence images of RV infection after treatment with live or γ-RV sera at 1:1280 dilution. DAPI channel (blue) indicates cell nuclei, and FITC channel (green) indicates RV infection. Scale bar = 100 μm.</p

RV-specific antibody responses.

<p>Mice were primed twice with live RV or γ-RV, 2 weeks apart. Serum samples harvested on Day 14 post-2^nd priming and analysed for RV-specific IgG using ELISA. (A) Serial dilutions of serum samples and absorbance readings at 450/620 nm for total IgG. (B) IgG titres in primed groups calculated relative to cut-off value (dotted line), determined using OD values of serum from control mice. Data presented as mean ± SEM (n = 6), and analysed by unpaired t-test (n.s., not significant) (C) Absorbance at 450/620nm for total IgG, IgG1, and IgG2c in serum at 1:200 dilution by ELISA. Data presented as mean ± SEM (n = 4), analysed by One-Way ANOVA (****, p < 0.0001).</p

Sterility testing of 50 kGy γ-irradiated RV.

<p>Monolayers of MA104 cells were incubated for 24h with live RV or γ-RV at 4 x 10⁵ FFU-equivalent/well. Culture supernatant was harvested and used to infect new MA104 cell monolayers, and previously infected monolayers stained for RV infection by FFA. DAPI channel (blue) indicates cell nuclei, and FITC channel (green) indicates RV infection. Stained monolayers visualised using Nikon TiE inverted fluorescence microscope. Scale bar = 100 μm. Images representative of 5 replicates per sample for each passage.</p