title: In vitro pyrogenicity of Gram-positive bacteria--validation of the kit using fresh human whole blood.
abstract: The two conventional tests to detect pyrogen contaminants in injectable pharmaceutical drugs are the Rabbit Model and the Limulus amoebocyte lysate (LAL) test. To replace these models, a new system on human whole blood is developed, using the release of Interleukin 1 beta (IL1beta) after cell stimulation with gram-positive and gram negative pyrogens. The purpose of this study was to validate the ENDOSAFE-IPT kit using the quantitative ELISA enzyme immunoassay. The assay is divided into two parts: blood cell stimulation with Lipopolysaccharides (LPS) and Lipoteichoic acid (LTA) and quantitation of IL1beta using the ELISA method. In each assay, blood from a particular donor were stimulated with the Endotoxin Standard, and with a sample of a commercial antibiotic preparation (Clavulanic acid/Ticarcillin) spiked with the Endotoxin Standard. LTA from Bacillus subtilis and a sample of diphtheria toxoid were also used. At least, six assays were tested. A polynomial regression of the Endotoxin Standard series showed a correlation coefficient greater than 0.99. The spiked antibiotic sample recoveries were 50-121%. The LTA quantitation limit was 0.1 microg/ml and the range of detection of pyrogens from Gram positive diphtheria toxoid was 0.77 to 2.5 EEU/ml. The IL1beta production varied markedly between donors. However the coefficient of variation was less than 20 % intra-assay. In conclusion, the ENDOSAFE-IPT kit can be used for the quantitative and qualitative detection of pyrogens from Gram negative and Gram positive bacteria.
authors: French Health Products Safety Agency (Afssaps French OMCL) DLC, CBR department, 635 rue de la Garenne, 34740 Vendargues, France.
authors: C
authors: C
authors: François
authors: J
authors: J
authors: Neveu
authors: D
authors: D
authors: Sauvaire
authors: P A
authors: PA
authors: Bonnet
authors: M H
authors: MH
authors: Tissier
mesh_terms: D001412:Bacillus subtilis / Q000472:pathogenicity; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D001769:Blood; D019818:Clavulanic Acid; D004168:Diphtheria Toxoid; D004340:Drug Contamination; D004797:Enzyme-Linked Immunosorbent Assay; D006090:Gram-Negative Bacteria / Q000472:pathogenicity; D006094:Gram-Positive Bacteria / Q000472:pathogenicity; D006801:Humans; D053583:Interleukin-1beta / Q000032:analysis / Q000378:metabolism*; D008070:Lipopolysaccharides; D004364:Pharmaceutical Preparations / Q000032:analysis*; D011749:Pyrogens / Q000032:analysis*; D011786:Quality Control; D015203:Reproducibility of Results; D013682:Teichoic Acids; D013982:Ticarcillin
chemical_list: D015415:Biomarkers; D004168:Diphtheria Toxoid; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D004364:Pharmaceutical Preparations; D011749:Pyrogens; D013682:Teichoic Acids; D019818:Clavulanic Acid; C009900:lipoteichoic acid; D013982:Ticarcillin