SENSITIVE QUANTITATION OF ENDOTOXIN BY ENZYME-LINKED-IMMUNOSORBENT-ASSAY WITH MONOCLONAL-ANTIBODY AGAINST LIMULUS PEPTIDE-C

Limulus peptide C, a 28-amino-acid fragment of coagulogen formed by th e reaction of endotoxin with Limulus amebocyte lysate, was synthesized , and a monoclonal antibody against it was raised. A new microassay fo r endotoxin was developed, using this antibody in an enzyme-linked imm unosorbent assay for generated peptide C-like immunoreactivity. A line ar relationship between absorbance and endotoxin concentration was obt ained. Control standard endotoxin in water could be detected to a leve l of 0.001 endotoxin unit per mi. The endotoxin levels in plasma sampl es from normal humans, rabbits, mice, and guinea pigs were generally f ound to be below the detection limit of 0.01 endotoxin unit per mi of plasma. The color and turbidity of specimens did not interfere with th e 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 tot ally unresponsive to natural glucans. The monoclonal antibody cross-re acted with peptide C-like immunoreactivity generated in Tachypleus ame bocyte lysate, which gave equal sensitivity in the endotoxin assay.