Sensitive quantitation of endotoxin by enzyme-linked immunosorbent assay with monoclonal antibody against Limulus peptide C

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Limulus peptide C, a 28-amino-acid fragment of coagulogen formed by the reaction of endotoxin with Limulus amebocyte lysate, was synthesized, and a monoclonal antibody against it was raised. A new microassay for endotoxin was developed, using this antibody in an enzyme-linked immunosorbent assay for generated peptide C-like immunoreactivity. A linear relationship between absorbance and endotoxin concentration was obtained. Control standard endotoxin in water could be detected to a level of 0.001 endotoxin unit per ml. The endotoxin levels in plasma samples from normal humans, rabbit, mice, and guinea pigs were generally found to be below the detection limit of 0.01 endotoxin unit per ml of plasma. The color and turbidity of specimens did not interfere with the assay. The consumption of Limulus amebocyte lysate in the assay was less than 5% of that in the gel-clot and chromogenic assays. With raw lysate, which was much more stable in solution than chloroform-treated lysate, the assay was still highly sensitive to endotoxin but was totally unresponsive to natural glucans. The monoclonal antibody cross-reacted with peptide C-like immunoreactivity generated in Tachypleus amebocyte lysate, which gave equal sensitivity in the endotoxin assay.

Original languageEnglish
JournalJournal of Clinical Microbiology
Volume32
Issue number2
Pages (from-to)416-22
Number of pages7
ISSN0095-1137
Publication statusPublished - Feb 1994

    Research areas

  • Animals, Antibodies, Monoclonal, Bacterial Toxins/analysis, Blood Proteins/immunology, Endotoxins/analysis, Enzyme-Linked Immunosorbent Assay/methods, Glucans, Horseshoe Crabs, Limulus Test/methods, Mice, Peptide Fragments/immunology, Reference Standards, Salmonella, Sensitivity and Specificity

ID: 203630401