Detection and quantitation of endotoxin by fluorescence polarization

A method of detecting endotoxin in a liquid sample suspected of containing endotoxin, comprising admixing said liquid sample with fluorescently labeled Endotoxin Neutralizing Protein (SEQ ID NO:3) for a time sufficient to form a complex between the labeled Endotoxin Neutralizing Protein (SEQ ID NO:3) and the endotoxin, and performing a fluorescence polarization experiment on said sample to obtain a fluorescence polarization value, wherein a change in the fluorescence polarization value after formation of the complex indicates the presence of endotoxin, wherein the fluorescence polarization experiment comprises:

• (a) inserting a control container containing buffer or water into a fluorescence polarization analyzer;
• (b) adding a predetermined amount of a standard solution of the fluorescently labeled Endotoxin Neutralizing Protein (SEQ ID NO:3) to the control container to obtain a solution, and measuring the fluorescence polarization to give a baseline measure of fluorescence polarization;
• (c) adding to the solution obtained in (b) a series of known concentrations of lipopolysaccharide to obtain a mixture, and recording any change in fluorescence polarization;
• and
• (d) adding to the solution obtained in (b) or to the mixture obtained in (c) the complex comprising fluorescently labeled Endotoxin Neutralizing Protein (SEQ ID NO:3) and endotoxin, and recording any change in fluorescence polarization.

A method of quantifying endotoxin in a liquid sample suspected of containing endotoxin, comprising admixing said liquid sample with fluorescently labeled Endotoxin Neutralizing Protein (SEQ ID NO:3) for a time sufficient to form a complex between the labeled Endotoxin Neutralizing Protein (SEQ ID NO:3) and the endotoxin, and performing a fluorescence polarization experiment on said sample, wherein any relative change in fluorescence polarization after the complex is formed is a measure of the quantity of endotoxin in said sample, wherein said fluorescence polarization experiment comprises:

• (a) inserting a control container containing buffer or water into a fluorescence polarization analyzer;
• (b) adding a predetermined amount of a standard solution comprising Endotoxin Neutralizing Protein (SEQ ID NO:3) to the control container to obtain a solution, and measuring the fluorescence polarization to give a baseline measure of fluorescence polarization;
• (c) adding to the solution obtained in (b) a series of known concentrations of lipopolysaccharide to obtain a mixture, recording any change in fluorescence polarization, and plotting the changes to give a standard curve;
• and
• (d) adding to the solution obtained in (b) or to the mixture obtained in (c) the complex comprising fluorescently labeled Endotoxin Neutralizing Protein (SEQ ID NO:3) and endotoxin, and recording any change in fluorescence polarization.

A method of detecting endotoxin in a liquid sample suspected of containing endotoxin, comprising:

• (a) admixing a defined concentration of Endotoxin Neutralizing Protein with a defined concentration of fluorescently labeled lipopolysaccharide for a time sufficient to form a complex between the labeled lipopolysaccharide and the Endotoxin Neutralizing Protein;
• (b) performing a fluorescence polarization experiment on the admixture obtained according to (a) to give a first fluorescence polarization value;
• (c) admixing a sample suspected of containing endotoxin with the admixture obtained in (a);
• and
• (d) performing a fluorescence polarization experiment on the admixture obtained in (c) to give a second fluorescence polarization value;
• wherein a change in the second fluorescence polarization value with respect to the first fluorescence polarization value indicates the presence of endotoxin.

A method of quantifying endotoxin in a liquid sample suspected of containing endotoxin, comprising:

• (a) admixing a defined concentration of Endotoxin Neutralizing Protein with a defined concentration of fluorescently labeled lipopolysaccharide for a time sufficient to form a complex between the labeled lipopolysaccharide and the Endotoxin Neutralizing Protein;
• (b) performing a fluorescence polarization experiment on the admixture obtained according to (a) to give a first fluorescence polarization value;
• (c) admixing a sample suspected of containing endotoxin to the admixture obtained in (a);
• and
• (d) performing a fluorescence polarization experiment on the admixture obtained in (c) to give a second fluorescence polarization value;
• wherein the relative change in the second fluorescence polarization value with respect to the first fluorescence polarization value is a measure of the quantity of endotoxin in said sample.