Abstract
The safe and effective dosing of botulinum neurotoxins (BoNTs) requires accurate and reliable methods to measure their potency. Several novel methods have been introduced over the past decade; however, only few studies have compared the potency of BoNT products with that of the LD50 and other alternative assays. Therefore, the objective of this study was to comparatively evaluate widely used BoNT products using various test methods. Four types of BoNTs (prabotulinumtoxin A, onabotulinumtoxin A, neubotulinumtoxin A, and letibotulinumtoxin A) were used in this study. The estimated potency was assessed using the LD50 assay, and the total BoNT type A protein levels were measured using the enzyme-linked immunosorbent assay (ELISA). The in vitro efficacy of the BoNTs was determined using fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) assays. The results showed differences in the total amount of BoNT protein and the cleavage activity of SNAP-25 within all types of BoNTs. The SPR study seemed to be useful for evaluating the potency by specifically measuring intact 19S neurotoxin, and these results provide new insights for assessing different BoNT products.
Highlights
The therapeutic and cosmetic applications of botulinum neurotoxins (BoNTs) have progressed steadily over the last 30 years since the FDA approved its use for the treatment of strabismus in 1989
The potency of each BoNT product was calculated based on the mouse LD50 units observed in each group, defined as the doses that cause 50% death in 10 mice within 72 h following intraperitoneal injection
Sandwich enzyme-linked immunosorbent assay (ELISA) was carried out to determine the total amounts of botulinum toxin proteins, either active or inactive, present in each vial of the indicated products, which is required to calculate the toxin efficacy value corresponding to the same toxin dose across the samples
Summary
The therapeutic and cosmetic applications of botulinum neurotoxins (BoNTs) have progressed steadily over the last 30 years since the FDA approved its use for the treatment of strabismus in 1989. BoNTs are produced by various Clostridium species and were classified into different serotypes based on their immunological properties. With the development of genomic sequencing techniques, novel BoNT serotypes and BoNTlike toxins from non-clostridial species have been discovered [1,2,3,4]. BoNTs exert their neurotoxic activity by blocking neuromuscular conduction via the inhibition of presynaptic release of acetylcholine into neuromuscular junction [5]. For BoNT products utilized for medical purposes, the biological activity of the active component must be accurately determined for each batch using suitable and highly precise assay systems. Due to its high sensitivity, the in vivo mouse LD50 assay was adopted by all manufacturers to evaluate the potency of BoNT [7]. It assesses the amount of toxin needed to kill 50% of the mice when the preparation is injected intraperitoneally
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