Improvements in the sensitivity and reproducibility of nephelometric endotoxin determinations using endotoxin-activated enzyme from Limulus amebocyte lysate are achieved by contacting endotoxin-coagulated lysate with an ionic surface active suspending agent, generally a mixture of detergent and suspending agent. Instrumentation improvements further enhance results.
A process for measuring the amount of endotoxin or (1.fwdarw.3)-.beta.-D-glucan (referred to hereinafter as .beta.-glucan) contained in a sample, which comprises mixing the sample with amebocyte lysate of horseshoe crabs in the presence of at least one water-soluble polymer selected from the group consisting of a polyethylene glycol, a polyvinyl alcohol, a methyl cellulose and a hydroxypropyl cellulose, applying a light to the resulting mixture, measuring the time required until a degree of optical variation of the resulting mixture reaches a predetermined value after the mixing of the sample with the amebocyte lysate, or after the lapse of the predetermined time from the mixing of the sample with the amebocyte lysate, and determining the amount of endotoxin or .beta.-glucan contained in the sample on the basis of the relationship between the said time and the amount of endotoxin or .beta.-glucan; and also a reagent for measuring the amount of endotoxin or .beta.-glucan contained in a sample, which comprises amebocyte lysate of horseshoe crabs and at least one water-soluble polymer selected from the group consisting of a polyethylene glycol, a polyvinyl alcohol, a methyl cellulose and a hydroxypropyl cellulose.
A pretreating method for a sample such as plasma for endotoxin measurement which includes diluting the sample with a surfactant-containing aqueous solution, and subjecting the diluted sample to heat treatment can prevent influences of inhibitors, etc. present in the sample and gives high recovery of endotoxins.
A p-nitrophenyl hydrolase contaminant present in Limulus coagulase sources interferes in, i.e., inhibits, coagulase-based assays for endotoxin. The contaminant inhibitor enzyme may be removed from coagulase by physical separation or by selective denaturation, for example by adjusting the pH of a coagulase solution to about from 8.5 to 11. Inhibitor-free coagulase is a novel composition. The coagulase activity of the composition is adjusted to a predetermined level to provide a standardized coagulase reagent for use in endotoxin assays.
The invention provides methods and compositions for the detection and/or quantification of bacterial endotoxins. In particular, provided herein is an inexpensive and reproducible method for producing an improved amebocyte lysate preparation having reduced Factor G activity. Provided also is an endotoxin-specific amebocyte lysate preparation produced by such a method. In addition, the invention provides methods and compositions akin for enhancing the sensitivity to endotoxins of amebocyte lysate preparations having reducing Factor G activity. In particular, the sensitivity of such amebocyte lysate preparations to endotoxins can be enhanced by the addition of exogenous (1.fwdarw.3) .beta.-D-glucan.
The invention provides methods and compositions for the detection and/or quantification of bacterial endotoxins. In particular, provided herein is an inexpensive and reproducible method for producing an improved amebocyte lysate preparation having reduced Factor G activity. Provided also is an endotoxin-specific amebocyte lysate preparation produced by such a method. In addition, the invention provides methods and compositions for enhancing the sensitivity to endotoxins of amebocyte lysate preparations having reducing Factor G activity. In particular, the sensitivity of such amebocyte lysate preparations to endotoxins can be enhanced by the addition of exogenous (1.fwdarw.3) .beta.-D-glucan.
