Intralaboratory validation of kinetic chromogenic Limulus amebocyte lysate assay for bacterial endotoxin determination in anti-bothropic serum

Abstract

Over the years, substituting in vitro biological methods for in vivo tests has posed an ever increasing challenge for researchers, including those who study the applications for snake antivenom. In the quality control of antivenons, the only official test recommended by pharmacopoeias for detecting pyrogenicity is the rabbit pyrogen test. In the present study, we propose intralaboratory validation of a method to replace the rabbit pyrogen test: in vitro determination of bacterial endotoxin in anti-bothropic serum (ABS) with quantitative kinetic chromogenic limulus amebocyte lysate (LAL) assay. The kinetic chromogenic LAL assay is specific to the detection of gram-negative bacterial endotoxin. The validation of the test involved the determination of performance parameters required by the Agência Nacional de Vigilância Sanitária Brasileira (ANVISA, the Brazilian National Health Surveillance Agency), the United States Food and Drug Administration (FDA) and the United States Pharmacopeia (USP) 35. In all experiments, the correlation coefficient of the curve obtained with the control standard endotoxin (CSE; Escherichia coli 055:B5 strain, range, 0.005–50EU/mL) was between −0.998 and −1.000; and the recovery of endotoxin added to the sample of ABS (0.5EU/mL) at the working dilution (1:10) followed the recuperation criteria (i.e., 50–200%). We performed six determinations, in each of which the coefficient of variation for the intermediate precision was between 5.6% and 13.8% (below the 15% threshold) and the accuracy was between 90.7% and 114.3% (within the acceptable range of 80–120%). The endotoxin concentration limit for the ABS was determined to be ≤2.9EU/mL. The intralaboratory validation of the methodology was considered to have been successful because it met the criteria for all of the performance parameters.