Limits of detections and quantification.

Pyrogens, which include endotoxin and non-endotoxin pyrogens (NEPs), act on immune cells in the bloodstream, causing various effects such as fever and endotoxic shock. The limulus amebocyte lysate test, a commonly used endotoxin test in the manufacturing of pharmaceuticals and medical devices, can detect endotoxin but not NEPs. The monocyte activation test (MAT), which uses monocytes, is a testing method included in the European Pharmacopoeia (EP 11.5; 07/2024:20630) that can detect NEPs. The MAT detects the cellular response following activation of Toll-like receptors (TLRs) by pyrogens; released cytokines, such as IL-6, are often the targets of detection. This cytokine release is regulated by the transcription factor NF-κB. In this study, we investigated whether it is possible to detect pyrogens with an NF-κB reporter gene-expressing cell line, using the NOMO-1 cell line as a model monocyte-like line. This study demonstrates that the reporter gene-expressing cells can detect 0.0125 EU/mL lipopolysaccharide (LPS) after 3 hours of incubation, and a stable calibration curve for LPS quantification can be created. Moreover, these cells can detect agonists for TLR1–9 in a concentration-dependent manner. Pharmaceuticals, including blood products and antibody drugs, were used in LPS recovery tests to confirm that they do not interfere with LPS detection. This study demonstrates that NF-κB reporter cells facilitate a simpler, more concise MAT, eliminating the complexity associated with enzyme-linked immunosorbent assays. Moreover, using the NOMO-1 cell line allows for the detection of a wider range of NEPs compared with using existing reporter gene-expressing cell lines.

热原（Pyrogens）包括内毒素（endotoxin）与非内毒素热原（non-endotoxin pyrogens, NEPs），可作用于血液中的免疫细胞，引发发热、内毒素休克等多种病理效应。鲎阿米巴样细胞溶解物试验（limulus amebocyte lysate test）是制药与医疗器械生产中常用的内毒素检测方法，仅可检出内毒素，无法识别NEPs。单核细胞活化试验（monocyte activation test, MAT）是收录于《欧洲药典》（European Pharmacopoeia, EP 11.5; 07/2024:20630）的检测方法，该方法以单核细胞为检测载体，可检出NEPs。MAT通过检测热原激活Toll样受体（Toll-like receptors, TLRs）后引发的细胞应答实现检测，常以释放的细胞因子（如白细胞介素6, IL-6）作为检测靶点。此类细胞因子的释放过程受转录因子NF-κB调控。
本研究以NOMO-1细胞系（NOMO-1 cell line）作为类单核细胞模型，探究表达NF-κB报告基因的细胞系能否用于热原检测。实验结果显示，该报告基因细胞经3小时孵育后，可检出0.0125 EU/mL的脂多糖（lipopolysaccharide, LPS），且可建立稳定的LPS定量校准曲线。此外，该细胞系可呈浓度依赖性地检出TLR1~9的受体激动剂。
为验证各类药品对LPS检测的干扰情况，本研究选取血液制品、抗体药物等多款药物开展LPS回收率实验，结果证实此类药品不会干扰LPS检测。
本研究证实，NF-κB报告细胞可简化传统单核细胞活化试验流程，规避酶联免疫吸附试验（enzyme-linked immunosorbent assays, ELISA）相关的操作复杂度；且相较于现有报告基因细胞系，采用NOMO-1细胞系可实现更广范围的NEPs检测。