Soo-Hyun Kim1, Sung-Beom Kim, Gi-Hyeok Yang, Chang-Hoon Rhee. 1. Microbial Toxin Research Institute, Medytox Inc., 641-4, Gak-ri, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea. crusader729@medy-tox.co.kr
Abstract
PURPOSE: We performed a prevalidation of the compound muscle action potential (CMAP) assay to determine the potency of botulinum neurotoxin type A (BoNT/A) with the aim of substituting for the mouse lethality test (LD₅₀), which is used for quality control. METHODS: Prevalidation experiments were performed to demonstrate the specificity, linearity, accuracy, precision, range, limit of quantitation (LOQ), and robustness of the assay. For specificity, toxin detection ability was determined in the presence of neutralizing antibodies (0.8 and 8 IU/mL). Linearity of this assay was determined by measuring CMAP amplitude using nine concentrations (n = 3) in the range of 1-100 U/mL (n = 3). Accuracy was assessed using five concentrations (n = 3) in the range of 4-40 U/mL. Intermediate precision was confirmed by analyzing individually prepared reagents on multiple days by one operator (n = 3). Different body weights (23-25 and 25-27 g) and measurement times (3-5 and 5-7 min) after anesthetic induction were tested to assess robustness. RESULTS: This assay might have BoNT/A specificity, based on the CMAP amplitude recovery using a concentration of neutralizing antibodies. The calibration curves were linear over the range of 2-40 U/mL (R² = 0.982). The accuracy of 14 determinations was within the range of 89.8-118.6% compared to the theoretical values among 15 determinations, except one (131.3%). Assay variability was acceptable with coefficients of variation of 4.3-14.4%. The range of quantification and the LOQ were 4-40 U/mL and 4 U/mL, respectively. Different body weights and measurement times after inducing anesthesia had no effect on CMAP amplitude. CONCLUSIONS: These results suggest that the mouse CMAP assay is an alternative method to the standard LD₅₀ potency test and meets the requirement of the three Rs (particularly refinement and reduction).
PURPOSE: We performed a prevalidation of the compound muscle action potential (CMAP) assay to determine the potency of botulinum neurotoxin type A (BoNT/A) with the aim of substituting for the mouse lethality test (LD₅₀), which is used for quality control. METHODS: Prevalidation experiments were performed to demonstrate the specificity, linearity, accuracy, precision, range, limit of quantitation (LOQ), and robustness of the assay. For specificity, toxin detection ability was determined in the presence of neutralizing antibodies (0.8 and 8 IU/mL). Linearity of this assay was determined by measuring CMAP amplitude using nine concentrations (n = 3) in the range of 1-100 U/mL (n = 3). Accuracy was assessed using five concentrations (n = 3) in the range of 4-40 U/mL. Intermediate precision was confirmed by analyzing individually prepared reagents on multiple days by one operator (n = 3). Different body weights (23-25 and 25-27 g) and measurement times (3-5 and 5-7 min) after anesthetic induction were tested to assess robustness. RESULTS: This assay might have BoNT/A specificity, based on the CMAP amplitude recovery using a concentration of neutralizing antibodies. The calibration curves were linear over the range of 2-40 U/mL (R² = 0.982). The accuracy of 14 determinations was within the range of 89.8-118.6% compared to the theoretical values among 15 determinations, except one (131.3%). Assay variability was acceptable with coefficients of variation of 4.3-14.4%. The range of quantification and the LOQ were 4-40 U/mL and 4 U/mL, respectively. Different body weights and measurement times after inducing anesthesia had no effect on CMAP amplitude. CONCLUSIONS: These results suggest that the mouse CMAP assay is an alternative method to the standard LD₅₀ potency test and meets the requirement of the three Rs (particularly refinement and reduction).