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. 2022 Jul 19;2022:2748962. doi: 10.1155/2022/2748962

Figure 4.

Figure 4

Analysis of inhibition of proteolytic activity and cytotoxicity of BjussuMP-II by anti-BjussuMP-II VHHs, and circular dichroism of anti-BjussuMP-II VHH61. (a) Proteolytic activity of BjussuMP-II on casein after exposure to a preincubated solution containing the toxin and the VHHs (anti-BjussuMP-II VHH47 and anti-BjussuMP-II VHH61) in different molar ratios. NC: negative control consisting of casein and buffer. PC: casein incubated with 10 μg of BjussuMP-II. Columns marked with an asterisk (∗) represent a statistical difference in relation to the positive control (∗p < 0.001). (b) Analysis of the inhibition of LDH release by tEND cells against exposure to BjussuMP-II by anti-BjussuMP-II VHH61. Cytotoxicity estimated by LDH release after 48 h of exposure to a preincubated solution containing BjussuMP-II and anti-BjussuMP-II VHH61 at a 1 : 5 ratio (w/w). NC: negative control consisting of untreated cells. Triton: cells treated with Triton X-100 at a concentration of 0.1%. Columns marked with an asterisk (∗) represent a statistical difference in relation to the negative control (∗∗∗p < 0.001 and ∗∗p < 0.01). Columns marked with a pound sign (#) represent a statistical difference between BjussuMP-II and BjussuMP-II+anti-BjussuMP-II VHH 61 (#p < 0.05). (c) Analysis of anti-BjussuMP-II VHH61 circular dichroism spectra collected in the 191-260 nm interval and thermal denaturation curve obtained at a wavelength of 202 nm during heating from 20 to 90°C. The measurements were done in a JASCO J-815 spectropolarimeter.