Development of in-vitro methods to detect neutralizing antibody against the light chain of botulinum toxin in secondary botulinum toxin treatment failure patients

Abstract

Botulinum toxin serotype A (BoNT/A) is the most common type of botulinum toxin used in clinical practice, particularly in aesthetic minimally invasive procedures. Secondary treatment failure (STF) of BONT/A has been observed in cosmetic indications as the use of BONT/A has increased. STF is thought to be caused by the production of neutralizing antibodies (NAbs) against the light chain of BoNT/A. The research aimed to develop the fluorescence resonance energy transfer (FRET) method to detect NAbs against the light chain of BoNT/A by demonstrating the potential role of SNAPtide substrate cleavage by BoNT/A-coated tosyl-activated Dynabeads™ in a FRET assay. The blood was collected from the BoNT/A naive group consisting of four stable individuals who had never undergone BoNT/A treatment. The BoNT/A tolerant group consisted of four stable individuals who had undergone BoNT/A treatment but were unable to respond. Only the patients with frontalis test positive were recruited into this study. The level of fluorescence after the SNAPtide substrate cleavage was measured. In the FRET assay, it was found that the 1:200 diluted serum had minimal proteolytic activity. 80937 beads/25 µl coated with 500 units of BoNT/A in 500 µl PBS solution could cleave the 5 µM SNAPTide substrate. The fluorescent intensity of SNAPTide was cleaved by BoNT/A-coated beads, and those were still lower than those of cleavage by 1000 units/ml of free BoNT/A in solution. Therefore, the optimal preparation for the FRET assay was proposed as dilution of serum at1:200. The SNAPTide substrate could be cleaved by BoNT/A-coated beads, but the conditions for coating the beads had to be improved for further FRET assays to detect NAbs against the light chain of BoNT/A. IMPLICATION OF THE THESIS: This study would help us establish a non-cellular in-vitro immunological technique for detecting NAbs against the light chain of BoNT/A in STF patients in routine clinical practice and providing a rapid diagnosis. Furthermore, it has the potential to substitute MPA or MHDA by providing a precise result and avoiding the use of animals in the study.