Development and validation of pyrogen determination in vitro based on THP1-BlueTM NF-κB reporter gene assay
WU Yan-lin
ZHANG Ming-lu
YANG Ze-an
CHEN Hong-yu
ZHANG Yuan
Abstract：Objective:To establish a method for detection of pyrogens using THP-1 cells with NF-κB reporter gene and SEAP activity,and investigate the feasibility and species applicability of the method in vitro.Methods:With reference to the guidelines of the general rules of ChP,9101 Guidelines for validation of analytical methods and 9301 Guidelines for application of injection safety checking method(with monocyte activation reaction assay),and using the national standard product of bacterial endotoxin as the benchmark,a methodological study was conducted on the THP1-BlueTM NF-κB reporter gene assay to examine its methodic feasibility in terms of appropriateness,precision,and durability.Precision and threshold were used as the judgment indicators for the method feasibility analysis.A study was also conducted on the applicability of the assay to different varieties.Results:According to 9301 Guidelines for monocyte activation reaction assay in ChP,the established method met the requirements of monocyte activation reaction assay,and the linear R2 was above 0.999.The lowest detection limit was calculated as 0.03 EU·mL-1,and the accuracy of the method was 101％and 106％for the recovery experiments using the solutions with endotoxin content of 0.5 and 1.0 EU·mL-1,respectively.The method was successfully used to study the suitability of human prothrombin complex,lyophilized human fibrinogen,influenza b,23-valent pneumonia and cowpox vaccines.Conclusion:The THP1-BlueTM NF-κB reporter gene method meets the requirements of monocyte activation assay and can be used for detection of pyrogenic substances in vitro.
Keywords：monocyte activation experimentpyrogen detectionTHP1-BlueTM NF-κBreporter gene
Publication Date：2024-11-30
Online Publishing Date：2026-08-14(First online date of this platform, not the publication date of the document)
Pages：7( 2331-2337 )