Mouse phrenic nerve (MPN) hemidiaphragm assay

The MPN assay was performed as described previously^{14, 55}. To limit the consumption of mice, the left and right phrenic nerve hemidiaphragms were excised from female mice of strain RjHan:NMRI (18–25 g, Janvier, St Berthevin Cedex, France) and placed in an organ bath containing 4 mL of Earle’s Balanced Salt Solution. The pH was adjusted to 7.4, and oxygen saturation was achieved by gassing with 95% O₂ and 5% CO₂. The phrenic nerve was continuously electro-stimulated at a frequency of 1 Hz via two ring electrodes. The pulse duration was 0.1 ms and the current was 25 mA to achieve maximal contraction amplitudes. Isometric contractions were recorded with a force transducer (Scaime, Annemasse, France) and the software VitroDat (Föhr Medical Instruments GmbH (FMI), Seeheim, Germany). The resting tension of the hemidiaphragm was approximately 10 mN. In each experiment, the preparation was first allowed to equilibrate for 15 min under control conditions. Then, the buffer was changed to 4 mL of Earle’s Balanced Salt Solution supplemented with 0.1% BSA and the BoNT/A1-VHH-containing solution. To determine neutralization by the VHH, a constant BoNT/A1 concentration of 1.63 pM yielding a paralysis time t_½ of 53.7 min in the absence of VHH was incubated for 15 min at 37 °C with increasing concentrations of VHH. The previously reported calibration curve determined for BoNT/A1 (y (BoNT/A1; 0.408/0.81/1.63 pM) = −16.062Ln(x) + 60.648, R² = 0.99)⁵⁶ was used to calculate the concentration of non-neutralized BoNT/A1. Difference of applied and non-neutralized BoNT/A1 calculates the amount of neutralization.