5.2. Immunogenicity Tests
The presence of reactive serum anti-drug antibodies to daxibotulinumtoxinA and RTP004 was assessed using validated ELISAs. Both ELISA methods detected all human IgG subclasses, IgA, IgE, IgM and to a lesser extent IgD. Briefly, serum samples including controls were diluted 1:50 in assay buffer (2% chicken serum, 0.05% Tween-20 in phosphate buffered saline [PBS]) and incubated on blocked antigen-coated microplate wells (either daxibotulinumtoxinA [Revance Therapeutics, Nashville, TN, USA] or RTP004 [Bachem Americas Inc., Torrance, CA, USA]) on a plate shaker at ambient temperature for approximately 1 h. After the plates were washed, (0.05% Tween-20 in PBS) bound antibodies were detected by incubation for 1 h with Protein-A/G labeled with horseradish peroxidase (Thermo Scientific, Waltham, MA, USA). The plates were washed again and briefly incubated with peroxidase substrate tetramethyl-benzidine (KPL, Milford, MA, USA). After termination of the enzymatic reaction with an acidic stop solution (1N HA three-tiered testing paradigm consistent with guidance by the US Food and Drug Administration was implemented for in-study testing [Samples confirmed to be positive for the presence of anti-daxibotulinumtoxinA binding antibodies were assessed for the presence of neutralizing antibodies by the validated mouse protection assay [