Endotoxin Testing of Allergen Extracts

Abstract

RATIONALE: While endotoxins are ubiquitous in nature, their role in the immunoresponse is controversial. This study was conducted to optimize an assay to measure endotoxins in allergen extracts (AE) and determine their concentrations in various AE.METHODS: A chromogenic kinetic assay was optimized to generate a log-log endotoxin standard curve (50 EU/mL - 0.05 EU/mL), which was utilized to calculate test concentrations, including positive and negative controls. Assay performance characteristics including linearity, precision, and accuracy were evaluated. Spike recovery experiments were performed with components typically contained in AE, i.e., 50% and 10% glycerin; 0.4% and 0.2% phenol, and with solutions containing these combined components. The AE analyzed were derived from Dermatophagoides pteronyssinus (N = 44), D. farinae (N = 35), pollen (N = 49), fungi (N = 18), and foods (N = 6). Non-parametric statistical analysis was performed.RESULTS: Negative control values were below 0.05 EU/mL. The standard curve correlation coefficient was >0.998. Assay precision and accuracy were 8.8% and 17.4%, respectively. 50% and 10% glycerin interfered in the test (average endotoxin spike recoveries: 0.0% and 24.3%, respectively). Interference was overcome by increasing sample dilutions. Endotoxins were selectively detected in most AE, with the greatest concentrations measured in D. farinae AE (median = 1,424 EU/mL) and the least in fungal AE (median = 1.2 EU/mL).CONCLUSIONS: An assay to measure endotoxins in AE has been optimized. Endotoxins are present in AE at various concentrations, generally at levels less than FDA proposed thresholds for parenteral exposure to endotoxins in humans (2,800 - 3,500 EU/70-kg person). RATIONALE: While endotoxins are ubiquitous in nature, their role in the immunoresponse is controversial. This study was conducted to optimize an assay to measure endotoxins in allergen extracts (AE) and determine their concentrations in various AE. METHODS: A chromogenic kinetic assay was optimized to generate a log-log endotoxin standard curve (50 EU/mL - 0.05 EU/mL), which was utilized to calculate test concentrations, including positive and negative controls. Assay performance characteristics including linearity, precision, and accuracy were evaluated. Spike recovery experiments were performed with components typically contained in AE, i.e., 50% and 10% glycerin; 0.4% and 0.2% phenol, and with solutions containing these combined components. The AE analyzed were derived from Dermatophagoides pteronyssinus (N = 44), D. farinae (N = 35), pollen (N = 49), fungi (N = 18), and foods (N = 6). Non-parametric statistical analysis was performed. RESULTS: Negative control values were below 0.05 EU/mL. The standard curve correlation coefficient was >0.998. Assay precision and accuracy were 8.8% and 17.4%, respectively. 50% and 10% glycerin interfered in the test (average endotoxin spike recoveries: 0.0% and 24.3%, respectively). Interference was overcome by increasing sample dilutions. Endotoxins were selectively detected in most AE, with the greatest concentrations measured in D. farinae AE (median = 1,424 EU/mL) and the least in fungal AE (median = 1.2 EU/mL). CONCLUSIONS: An assay to measure endotoxins in AE has been optimized. Endotoxins are present in AE at various concentrations, generally at levels less than FDA proposed thresholds for parenteral exposure to endotoxins in humans (2,800 - 3,500 EU/70-kg person).