A Novel Reporter Gene Assay for Pyrogen Detection

He, Qing; Yu, Chuanfei; Wang, Lan; Ni, Yongbo; Zhang, Heng; Du, Ying; Gao, Hua; Wang, Junzhi

doi:10.7883/yoken.jjid.2019.163

Cited by 4 publications

(1 citation statement)

References 23 publications

(15 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Both RPT and MAT are not convenient. 33 , 34 To establish an in vitro model that is easy to use, we constructed a transgenic cell line by transfecting a luciferase reporter gene regulated by NF-κB into the human monocyte line THP-1, 35 , 36 which was screened for the NF-κB response by treatment with serial concentrations of LPS. The suitable subclone was selected mainly based on the signal-to-noise ratio (SNR) of the subclone’s NF-κB response, which was calculated as the subclone’s NF-κB response to LPS divided by that of the negative control.…”

Section: Resultsmentioning

confidence: 99%

A novel alternative for pyrogen detection based on a transgenic cell line

He,

Yu,

et al. 2024

Sig Transduct Target Ther

Self Cite

View full text Add to dashboard Cite

Pyrogen, often as a contaminant, is a key indicator affecting the safety of almost all parenteral drugs (including biologicals, chemicals, traditional Chinese medicines and medical devices). It has become a goal to completely replace the in vivo rabbit pyrogen test by using the in vitro pyrogen test based on the promoted ‘reduction, replacement and refinement’ principle, which has been highly considered by regulatory agencies from different countries. We used NF-κB, a central signalling molecule mediating inflammatory responses, as a pyrogenic marker and the monocyte line THP-1 transfected with a luciferase reporter gene regulated by NF-κB as an in vitro model to detect pyrogens by measuring the intensity of a fluorescence signal. Here, we show that this test can quantitatively and sensitively detect endotoxin (lipopolysaccharide from different strains) and nonendotoxin (lipoteichoic acid, zymosan, peptidoglycan, lectin and glucan), has good stability in terms of NF-κB activity and cell phenotypes at 39 cell passages and can be applied to detect pyrogens in biologicals (group A & C meningococcal polysaccharide vaccine; basiliximab; rabies vaccine (Vero cells) for human use, freeze-dried; Japanese encephalitis vaccine (Vero cells), inactivated; insulin aspart injection; human albumin; recombinant human erythropoietin injection (CHO Cell)). The within-laboratory reproducibility of the test in three independent laboratories was 85%, 80% and 80% and the interlaboratory reproducibility among laboratories was 83.3%, 95.6% and 86.7%. The sensitivity (true positive rate) and specificity (true negative rate) of the test were 89.9% and 90.9%, respectively. In summary, the test provides a novel alternative for pyrogen detection.

show abstract