Abstract
A new reliable treatment was developed for quantitative bacterial endotoxin microassay in rabbit blood, using limulus amebocyte lysate and chromogenic substrate (Boc-LeuGly-Arg-p-nitroanilide) . Some difficulties have occured in the use of the limulus amebocytelysate test, because of the presence of false negative and false positive substances which interfere with the limulus-endotoxin reaction in plasma. Optimal conditions to remove interferingsubstances in plasma samples prior to assay were determined. Addition of 0.26M perchloricacid to platelet-rich plasma and heated to 60°C for 5 min to remove interfering substances. Therecovery of endotoxins from various sources such as E. coli 0111: B4, E. coli 055: B5, E. coli O127: B8, S. typhimurium, S. minnesota 9700, S. tyhosa, S. abortus equi, S. enteritidis, Sigella flexneri, Pseudomonas aeruginosa was 103.3±4.9, 98.6±5.2, 109.7±5.9, 105.6±4.8, 100.5±4.7, 108.3±5.1, 105.2±4.4, 94.2±4.6, 96.9±3.7, and 102.1±5.2% (mean±SD; n=6), respectively. The result showed that the recoveries of various endotoxins from plasma werealmost 100%. The lower limit of assay sensitivity was 1pg/ml.
The widely used chloroform extraction method was found not to be effective to remove theinterefering substances from plasma, the recovery was 6.9±2.0% (mean±SD ; n=5) . The dilution and heating extraction method was found to be effective to remove false positive substances, but endotoxin recovery from plasma was poor.
The endotoxin concentration in plasma of rabbits after intravenous injection was assayed.As a result, in the group receiving 5μg/kg of endotoxin, approximately 98%of the endotoxin disappeared within 30min. However, blood specimens in the group receiving 500μg/kg reachedequilibrium in vivo within 15 min and two of three rabbits were killed. The high level ofendotoxin remained in the plasma of survived rabbit 24hr after receiving 500μg/kg of endotoxin.
The pyrogenicity of ndotoxin was found not to be the direct action. The pyrogenicity seemed to be the action by the endogenious pyrogen.
