<i>Ex vivo</i> mouse phrenic nerve-hemidiaphragm assay with ELC18.

<p>A) Neutralization of BoNT/E3 holotoxin (330 pg/mL; 20LD₅₀/mL) by different concentrations of ELC18. Neutralization properties of ELC18 as scFv at 165 nM, 16.5 nM, 3.3 nM, and 0.3 nM were evaluated. B) Neutralization properties of ELC18 as scFv-Fc at 9.4 nM, 4.7 nM, and 0.9 nM were evaluated. Each neutralization set is from a single hemidiaphragm preparation per concentration.</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

<i>In vivo</i> mouse paralysis assay with ELC18 as scFv-Fc.

<p>A) The neutralization activity of ELC18 was determined in the mouse paralysis assay <i>in vivo</i>. BoNT/E3 holotoxin (1LD₅₀ per dose) was pre-mixed with each antibody at 0.064 ng, 0.32 ng, 1.6 ng, 8 ng, 40 ng, 0.2 μg and 1 μg per dose. Animals were scored at 24 h post injection. Results are expressed as mean score for 4 mice ± SEM. Positive control group of mice were injected with BoNT/E3 toxin alone and negative control group of mice received the maximum concentration of antibody in the absence of toxin. B) Schema of the <i>in vivo</i> mouse paralysis assay. Formation of abdominal ptosis after injection of botulinum neurotoxin.</p

<i>Ex vivo</i> phrenic nerve-hemidiaphragm assay.

<p>Neutralization of BoNT/E3 toxicity (20LD₅₀/mL) by the BoNT/E3 specific scFv ELC18, ELC51 and ELC76.</p

Schema of macaque immunization.

<p>Immunization was performed with five subcutaneous injections of recombinant BoNT/E3 light chain and sera were sampled to estimate the immunization titer.</p

Inhibition of BoNT/E3 endopeptidase activity.

<p>Inhibition of BoNT/E3 endopeptidase activity.</p

<i>In vitro</i> endopeptidase assay.

<p>Concentration-dependent inhibition of the endopeptidase activity of BoNT/E3 holotoxin (330 pg/mL; 20LD₅₀/mL) <i>in vitro</i> by several scFv targeting the light chain of BoNT/E3. As control, SNAP–25 cleavage by BoNT/E3 without inhibiting scFv and buffer without toxin. Each data point is a mean of triplicate determinants from two separate experiments (n = 6) ± SEM.</p

Affinities of the four inhibiting scFv.

<p>Affinities of the four inhibiting scFv.</p

Antigen ELISA of the humanized variants of SEM120-IIIC1 against recombinant light chain of BoNT/A1.

<p>Binding of the germline-humanized anti-BoNT/A1 antibodies (hu1-hu16SEM120-IIIC1) as scFv-Fc (each 1 μg) was tested on 100 ng recombinant BoNT/A1 light chain.</p

GI value of the humanized variants of the different humanized variants of BLC7 and B2-7.

<p>GI value of the humanized variants of the different humanized variants of BLC7 and B2-7.</p