Assays for endotoxin
AssigneeNational University of Singapore
InventorsJeak Ling Ding, Bow Ho
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Abstract
The horseshoe crab, Carcinoscorpius rotundicauda Factor C cDNA (CrFC21) has been cloned into a shuttle baculoviral vector and another vector suitable for expression in insect cells. The recombinant baculoviral DNA was then transfected into the insect cells for expression of recombinant Factor C. Recombinant Factor C was found to be immunoreactive and is capable of binding both free and bound/immobilized lipid A. It is enzymatically active when triggered by LPS. The rFC is probably of the two-chain form, being cleaved into the heavy and light chains after activation by Gram negative bacterial endotoxin. As low as 0.01 pg (0.001 ng/ml) of LPS was detectable by the rFC, thus, indicating its potentials as a novel generation of “limulus amoebocyte lysate.”
Claims25
An assay for endotoxin, said assay comprising:
- a) contacting a sample to be assayed, wherein said sample comprises a peptide cleavable to produce a chromogenic or fluorogenic moiety, with a recombinant Factor C of a horseshoe crab, wherein said recombinant Factor C is obtained from an insect host cell culture and has protease activity;
- and
- b) measuring the amount of said chromogenic or fluorogenic moiety cleaved from said peptide, wherein cleavage of said moiety from said peptide is indicative of the presence of said endotoxin in said sample.
An assay for endotoxin, said assay comprising:
- a) contacting a sample to be assayed, said sample having a volume of about 200 μl and comprising a peptide cleavable to produce a chromogenic or fluorogenic moiety, with a recombinant Factor C of a horseshoe crab, wherein said recombinant Factor C is produced using a vector comprising a cDNA encoding Factor C, is obtained from an insect host cell culture, and has protease activity;
- and
- b) measuring the amount of said chromogenic or fluorogenic moiety cleaved from said peptide, wherein cleavage of said moiety from said peptide is indicative of the presence of said endotoxin in said sample.
An assay for endotoxin, said assay compnsing:
- a) mixing a sample to be assayed with a recombinant Factor C of a horseshoe crab, wherein said recombinant Factor C is obtained from an insect host cell culture and has protease activity, and incubating the mixed sample;
- b) adding to the incubated, mixed sample a peptid that is cleavable to produce a chromogenic or fluorogenic moiety and continuing incubation to allow cleavage of the peptide by Factor C activated by endotoxin in said sample;
- and
- c) measuring the amount of said chromogenic or fluorogenic moiety cleaved from said peptide, wherein cleavage of said moiety from said peptide is indicative of the presence of said endotoxin in said sample.
Dependencies are inferred from the claim text.