Journal: Scientific Reports
Article Title: An in vitro methodology for discovering broadly-neutralizing monoclonal antibodies
doi: 10.1038/s41598-020-67654-7
Figure Lengend Snippet: Binding and neutralizing capability of the scFv clones discovered using phage display and a cross-panning strategy against CTxs from African cobras. ( a ) In direct ELISA, 113 cross-binding scFv clones recognized both Nn 18 and Nn 20 venom fractions. Fraction 8 from Dendroaspis polylepis venom (Dp 8, Dendrotoxin 1) was used as a negative control. ( b ) Seven of the unique cross-binding scFv clones bound five different CTxs-containing fractions from three different cobra species (Nn 18, Nn 20, Nn 25, Nmo 13, and Nm 17). Bars represent mean ± SD of triplicate wells. ( c ) Different amounts of venom fractions (2–80 µg/mL) were added to N/TERT cells. After 24 h of incubation at 37 °C, cell viability was determined by quantifying the ATP release into the culture media, and mean cell survival percentage was calculated from triplicate wells. The half-maximal inhibitory concentration (IC 50 ) was determined by fitting the mean cell survival percentage of inhibition-concentration data into a Hill equation and has been represented as values ± standard error. ( d ) Neutralization of the cytolytic effects of the venom fractions on human N/TERT cells by the discovered broadly-neutralizing TPL0027_01_F7 scFv antibody. 2 IC 50 of the venom fractions were pre-incubated with TPL0027_01_F7. The scFv antibody TPL0039_05_E2, targeting the unrelated Myotoxin II from Bothrops asper venom, was used as a negative control. Experiments were performed in triplicates, and results are expressed as mean ± SD. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was performed for each group, except the Nn 18 group, and p < 0.05 was considered to be of statistical significance. For the Nn 18 group, a one-way ANOVA could not be carried out because the group only contained two samples, therefore, an unpaired t test was carried out with p < 0.05 considered to be of statistical significance. For each of the five fractions, there was a statistically significant difference between “fraction + TPL0027_01_F7” and “fraction”. For the four groups containing the “fraction + TPL0035_09_E2”, there was a statistically significant difference between this and “fraction + TPL0027_01_F7”. However, differences between “fraction” and “fraction + TPL0035_09_E2” were not statistically significant for any of the four groups. *Insufficient amounts, this fraction was not available for testing.
Article Snippet: The IONTAS human single-chain variable fragment (scFv) antibody phage library containing a clonal diversity of 4 × 10 10 was employed for phage display selections.
Techniques: Binding Assay, Clone Assay, Direct ELISA, Negative Control, Combined Bisulfite Restriction Analysis Assay, Incubation, Concentration Assay, Inhibition, Neutralization
