In vitro pyrogenicity of Gram-positive bacteria--validation of the kit using fresh human whole blood.
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.
François
C
C
French Health Products Safety Agency (Afssaps French OMCL) DLC, CBR department, 635 rue de la Garenne, 34740 Vendargues, France.
Neveu
J
J
Sauvaire
D
D
Bonnet
P A
PA
Tissier
M H
MH
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
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