Biacore experiment to evaluate the reactivity between anti-LF polyclonal antibodies and the two LF variants, in which LF(178–184) or LF (231–236) had been shaved in alanine.

<p>A CM5 chip was sensitized using LF variants in which LF(178–184) (blue) or LF (231–236) (red) had been shaved in alanine, as these variants did not react with scFv 2LF and their presence and general conformation had to be verified. The diluted (1:100) serum of a macaque, drawn before immunization with LF,was used as a negative control and did not react with these two LF variants (dashed curves). The serum from the same animal after hyper-immunization with LF reacted with these two LF variants (continuous curves), showing that they had been immobilized on the CM5 chip. The curves are representative of two separate Biacore experiments, realized in the same conditions except for the variants used for sensitization.</p

Mean identity between each epitope candidate in LF and the homologous region in EF, and mean solvent exposure of each epitope candidate.

<p>The identities between EF and LF epitope candidates are higher than the threshold, equal to 35%. The calculated mean solvent exposure for the five epitope candidates is 48%.</p

Biacore experiment to evaluate the reactivity between scFv 2LF and the five LF variants, in which H229, R230, Q234, L235 or Y236 had been individually mutated in alanine, or the LF variant where these five residues had been simultaneously mutated in alanine.

<p>A CM5 chip was sensitized with scFv 2LF. LF (control, in grey) and its five variants in which H229 (yellow), R230 (blue), Q234 (red), L235 (green) or Y236 (brown) had been individually mutated in alanine reacted in flux. The LF variant in which these five positions had been simultaneously mutated in alanine is presented in blue. Dilutions of LF and its variants giving an equivalent maximal signal are presented, to allow the comparison of the dissociation phases (after the maximal signal and the artefactual spikes) when dissociation constants are measured (between the 500^th and 700^th seconds). Values were calculated on several curves for each variant, and are presented on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065855#pone-0065855-t002" target="_blank">table 2:</a> the five variants with punctual mutations present at least a three-fold more rapid dissociation than the control, and the variant with the five simultaneous mutations presents no reactivity.</p

Biacore experiment to measure the affinity of scFv 2LF for EF.

<p>Sensorgrams were obtained at EF concentrations of 220 nM (red curve), 110 nM (light blue), 55 nM (dark blue), 28 nM (brown) and 14 nM (green) on a CM5 chip sensitized by 250 response units of scFv 2LF. The affinity of scFv 2LF for EF was measured as 5 nM.</p

IgG 2LF inhibits the formation of edema induced by the edema toxin (ET).

<p>(A) Photographs taken 12 hours after injection in mouse footpads of 20 µg ET (left picture), or of 20 µg ET pre-mixed with 5 µg (middle picture) or with 10 µg (right picture) of IgG 2LF. Ten µg of IgG 2LF premixed with 20 µg ET completely inhibited edema formation, whereas 5 µg of IgG 2LF reduced both the size and the duration of the edema; 2 µg of IgG 2LF had no appreciable effect. (B) Evolution of the sizes (dorsal/plantar) of mouse footpads after injection of 20 µg ET (blue), or of 20 µg ET pre-mixed with 2 µg (red) or 5 µg (yellow) or 10 µg (light blue) of IgG 2LF. Injection of PBS only (purple) was used as a negative control. Curves represent triplicate measurements for each timepoint. Pre-mixing with 10 µg of IgG 2LF completely abolished the formation of edema by 20 µg ET, and pre-mixing with 5 µg of IgG 2LF limited the size of the edema and reduced its duration. Pre-mixing of 20 µg ET with 2 µg of IgG 2LF had no appreciable effect.</p

Alignment of the sequences of LF_N and EF_N, and prediction of their secondary structures according to the LF 3D-structure (1J7N).

<p>The α helixes (1 to 12) are colored in red and β strands (1 to 4) in yellow. The residues that are identical, very similar and different between LF_N and EF_N are highlighted in the EF_N sequence in dark gray, light gray and white, respectively. The five linear regions identified as epitope candidates are indicated in blue, underlined and numbered: region 1 is LF(97–103), region 2 is LF(136–143), region 3 is LF(178–184), region 4 is LF(227–231) and region 5 is LF(232–236). The residues of LF_N constituting the scFv 2LF epitope are indicated in bold.</p

Evaluation of the reactivity of scFv 2LF with EF by ELISA and western blot.

<p>A/ Reactivity of scFv 2LF with EF in ELISA. Reactivity of scFv 2LF with EF is represented in blue, and LF (red) and KLH (green) were used as positive and negative controls, respectively. ScFv 2LF reacted with EF, though less strongly than with LF at high scFv concentrations. B/ Reactivity of scFv 2LF with EF in western blots under reducing conditions (lower part). EF (5, 2 and 1 µg/ml in lanes 4, 5 and 6, respectively) and LF as a positive control (5, 2 and 1 µg/ml in lanes 1, 2 and 3, respectively) were separated by SDS-PAGE electrophoresis (upper part) and their sizes verified to be 90 kDa by separation under reducing conditions. ScFv 2LF reacted similarly with EF and LF under reducing conditions.</p

ScFv 2LF dissociation rate constants with each LF variant, measured by Biacore experiments.

<p>The five residues whose mutation to alanine resulted in a 3-fold, or larger, decrease of the dissociation constant are shown in bold.</p

Results with web-based or stand-alone programs predicting 2LF epitopes.

<p>Results of 12 prediction methods used to predict 2LF epitopes are given as letters indicating the predictions and the method (a: BEPITOPE (TURNEE); b: BEPITOPE (TURN33); c: BepiPred; e: Ellipro; g: LEP-LP; h: BCPREDS; j: Epitopia; k: COBEpro; d: CBTOPE; f: Ellipro; i: DiscoTope; l: BEpro). The residues predicted as being part of the epitope are indicated by an “X” above the LF_N and EF_N alignment. The five epitope candidates identified by the <i>in silico</i> method developed in the present study are indicated with hyphens and numbered.</p

Human germline genes most similar to the genes encoding the BoNT/E3 specific scFv.

<p>Human germline genes most similar to the genes encoding the BoNT/E3 specific scFv.</p