title: Determination of Interference During In Vitro Pyrogen Detection: Development and Characterization of a Cell-Based Assay.
abstract: Contamination of pharmaceutical products and medical devices with pyrogens such as endotoxins is the most common cause of systemic inflammation and, in worst cases, of septic shock. Thus, quantification of pyrogens is crucial. The limulus amebocyte lysate (LAL)-based assays are the reference tests for in vitro endotoxin detection, in association with the in vivo rabbit pyrogen test (RPT), according to European Pharmacopoeia (EP 2.6.14), and U.S. Pharmacopoeia (USP <85>). However, several substances interfere with LAL assay, while RPT is not accurate, not quantitative, and raises ethical limits. Biological assays, as monocyte activation tests, have been developed and included in European Pharmacopoeia (EP 7.0; 04/2010:20630) guidelines as an alternative to RPT and proved relevant to the febrile reaction in vivo. Because this reaction is carried out by endogenous mediators under the transcriptional control of nuclear factor-kappaB (NF-kappaB), we sought to determine whether a NF-kappaB reporter-gene assay, based on MonoMac-6 (MM6) cells, could reconcile the basic mechanism of innate immune response with the relevance of monocytoid cell lines to the organism reaction to endotoxins. This article describes both optimization and characterization of the reporter cells-based assay, which overall proved the linearity, accuracy, and precision of the test, and demonstrated the sensitivity of the assay to 0.24 EU/mL endotoxin, close to the pyrogenic threshold in humans. Moreover, the assay was experimentally compared to the LAL test in the evaluation of selected interfering samples. The good performance of the MM6 reporter test demonstrates the suitability of this assay to evaluate interfering or false-positive samples.
authors.affiliation: 1 Department of Biomolecular Sciences, Section of Biochemistry and Molecular Biology, University of Urbino "Carlo Bo ," Urbino, Italy .
authors.forename: Linda
authors.initials: L
authors.lastname: Palma
authors.affiliation: 2 MolMed S.p.A. , Milan, Italy .
authors.forename: Francesca
authors.initials: F
authors.lastname: Rossetti
authors.affiliation: 1 Department of Biomolecular Sciences, Section of Biochemistry and Molecular Biology, University of Urbino "Carlo Bo ," Urbino, Italy .
authors.forename: Sabrina
authors.initials: S
authors.lastname: Dominici
authors.affiliation: 1 Department of Biomolecular Sciences, Section of Biochemistry and Molecular Biology, University of Urbino "Carlo Bo ," Urbino, Italy .
authors.forename: Costantina
authors.initials: C
authors.lastname: Buondelmonte
authors.affiliation: 3 Department of Biomolecular Sciences, Section of Pharmacology and Pharmacognosy, University of Urbino "Carlo Bo ," Urbino, Italy .
authors.forename: Marco B L
authors.initials: MB
authors.lastname: Rocchi
authors.affiliation: 2 MolMed S.p.A. , Milan, Italy .
authors.forename: Gian P
authors.initials: GP
authors.lastname: Rizzardi
authors.affiliation: 2 MolMed S.p.A. , Milan, Italy .
authors.forename: Giuliana
authors.initials: G
authors.lastname: Vallanti
authors.affiliation: 1 Department of Biomolecular Sciences, Section of Biochemistry and Molecular Biology, University of Urbino "Carlo Bo ," Urbino, Italy .
authors.forename: Mauro
authors.initials: M
authors.lastname: Magnani
mesh_terms: D016477:Artifacts; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D006801:Humans; D008070:Lipopolysaccharides; D009000:Monocytes / Q000187:drug effects* / Q000276:immunology*; D011749:Pyrogens / Q000008:administration & dosage* / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D008070:Lipopolysaccharides; D011749:Pyrogens
keywords: CD14; MonoMac-6; NF-kappaB; endotoxin; lipopolysaccharide; pyrogens