title: A rapid 'one-plate' in vitro test for pyrogens.
abstract: A rapid, 'one-plate' monocyte-activation test is described for detecting endotoxin and non-endotoxin pyrogens in parenteral medicinal products. The one-plate test offers useful gains over conventional 'two-plate' (cell culture plate+ELISA plate) tests in terms of its limit of detection, robustness, speed and cost. The 'one-plate' test is likely to be applicable to a wide range of products because it allows less time for product interference in the test. The 'one-plate' test utilises pyrogen-free anti-cytokine (interleukin (IL)-6 or tumour necrosis factor alpha (TNFalpha)) antibodies (Ab), coated and stabilised onto (pyrogen-free) 96-well plates. Monocytes/monocytic cells, endotoxin (lipopolysaccharides, LPS) standard or sample and (pyrogen-free) second (labelled) Ab are cultured together (usually for 2-4 h) on the Ab-coated plate and then the plate is washed and the ELISA completed. There is no transfer from one plate to another and no (further) incubations of (released) cytokine with, first, coating Ab and, then, developing Ab since these steps have already taken place during the initial cell culture. The rapid, 'one-plate' test is readily automated. The preferred readout is IL-6, which gives a limit of detection of 0.015 endotoxin units (EU)/ml with peripheral blood mononuclear cell (PBMNC), 0.03 EU/ml with diluted whole blood and 0.05 EU/ml with a monocytic cell line (MONO MAC 6).
authors.affiliation: National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, England, UK. spoole@nibsc.ac.uk
authors.forename: Stephen
authors.initials: S
authors.lastname: Poole
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Mistry
authors.forename: Christine
authors.initials: C
authors.lastname: Ball
authors.forename: Rose E
authors.initials: RE
authors.lastname: Gaines Das
authors.forename: Lisa Pieti
authors.initials: LP
authors.lastname: Opie
authors.forename: Garry
authors.initials: G
authors.lastname: Tucker
authors.forename: Mehul
authors.initials: M
authors.lastname: Patel
mesh_terms: D000328:Adult; D002460:Cell Line; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D015850:Interleukin-6 / Q000032:analysis; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D009000:Monocytes / Q000187:drug effects / Q000276:immunology; D011749:Pyrogens / Q000032:analysis* / Q000494:pharmacology; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
chemical_list: D016207:Cytokines; D015850:Interleukin-6; D008070:Lipopolysaccharides; D011749:Pyrogens; D014409:Tumor Necrosis Factor-alpha