Fever is a systemic inflammatory response of the body to pyrogens. Nuclear factor κB (NF-κB) is a central signaling molecule that causes the excessive secretion of various pyrogen-induced pro-inflammatory factors. This study explored the feasibility of a novel reporter gene assay (RGA) for pyrogen detection using RAW264.7 cells stably transfected with the NF-κB reporter gene as a pyrogenic marker. The RGA could detect different types of pyrogens, including the lipopolysaccharide of gram-negative bacteria, the lipoteichoic acid of gram-positive bacteria, and the zymosan of fungi, and a good dose-effect relationship was observed in terms of NF-κB activity. The limits of detection of the RGA to those pyrogens were 0.03 EU/ml, 0.001 μg/ml, and 1 μg/ml, respectively. The method had good precision and accuracy and could be applied to many molecules (e.g., nivolumab, rituximab, bevacizumab, etanercept, basiliximab, Haemophilus influenzae type b conjugate vaccine, 23-valent pneumococcal polysaccharide vaccine, group A and group C meningococcal conjugate vaccine, diphtheria, tetanus, pertussis [acellular, component], poliomyelitis [inactivated] vaccine, and imject alum adjuvant). The results of this study suggest that the novel RGA has a wide pyrogen detection spectrum and is sufficiently sensitive, stable, and accurate for various applications.
Keywords: Lipopolysaccharide; RAW264.7; fever; nuclear factor kappa B; pyrogens.