Method of determining the presence of endotoxin in a sample
First Claim
1. A method of determining the presence of an endotoxin or an endotoxin-like material in a sample, wherein the endotoxin-like material is selected from the group consisting of lipopolysaccharide, oligolipopolysaccharide, lipid A, glucans, peptidoglycan, lipoteichoic acid, lipoglycans and synthetic lipid A, the method comprising:
- (a) incubating a sample with a component which is from horseshoe crab amoebocyte lysate, hemolymph or a synthetic analogue thereof having substantially the same reactivity with an endotoxin or endotoxin-like material as a component occurring naturally in horseshoe crab amoebocyte lysate or haemolymph, whereby the properties of said component are altered if any endotoxin or endotoxin-like material is present in the sample so that no reaction with an antibody raised against or directed substantially only against said component or an immunological determinant thereof will take place,(b) reacting the incubated mixture of said sample and said component from step (a) with a monoclonal antibody raised against or directed substantially only against said component or an immunological determinant thereof whereby the antibody is bound to any of said component present in the mixture, and(c) determining the presence of any endotoxin or endotoxin-like material in the sample by detecting any bound antibody in the reaction mixture resulting from step (b), the detection of decreasing amounts of bound antibody showing the presence of an endotoxin or endotoxin-like material in the sample;
with the proviso that any of said component present after the incubation of the sample in step (a) is coupled to a solid support, orthat said antibody is coupled to a solid support or to a bridging molecule which is coupled to a solid support, orthat any endotoxin or endotoxin-like material present in the sample is coupled to a solid support.
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Abstract
In a method of determining the presence of an endotoxin or endotoxin-like material in a sample, a) a sample is incubated with a component of horseshoe crab amoebocytes lysate or haemolymph or a synthetic analogue thereof, b) the incubated mixture of the sample and the component or analogue resulting from step a) is reacted with an antibody raised against the component or analogue or against a reaction product of the incubation of step a), and c) the presence of endotoxin or endotoxin-like material in the sample is determined by detecting any bound antibody in the reaction mixture of step b). In the method either the component or analogue or the antibody or the endotoxin or endotoxin-like material is coupled to a solid support.
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Citations
29 Claims
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1. A method of determining the presence of an endotoxin or an endotoxin-like material in a sample, wherein the endotoxin-like material is selected from the group consisting of lipopolysaccharide, oligolipopolysaccharide, lipid A, glucans, peptidoglycan, lipoteichoic acid, lipoglycans and synthetic lipid A, the method comprising:
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(a) incubating a sample with a component which is from horseshoe crab amoebocyte lysate, hemolymph or a synthetic analogue thereof having substantially the same reactivity with an endotoxin or endotoxin-like material as a component occurring naturally in horseshoe crab amoebocyte lysate or haemolymph, whereby the properties of said component are altered if any endotoxin or endotoxin-like material is present in the sample so that no reaction with an antibody raised against or directed substantially only against said component or an immunological determinant thereof will take place, (b) reacting the incubated mixture of said sample and said component from step (a) with a monoclonal antibody raised against or directed substantially only against said component or an immunological determinant thereof whereby the antibody is bound to any of said component present in the mixture, and (c) determining the presence of any endotoxin or endotoxin-like material in the sample by detecting any bound antibody in the reaction mixture resulting from step (b), the detection of decreasing amounts of bound antibody showing the presence of an endotoxin or endotoxin-like material in the sample; with the proviso that any of said component present after the incubation of the sample in step (a) is coupled to a solid support, or that said antibody is coupled to a solid support or to a bridging molecule which is coupled to a solid support, or that any endotoxin or endotoxin-like material present in the sample is coupled to a solid support. - View Dependent Claims (3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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2. A method of determining the presence of an endotoxin or endotoxin-like material in a sample, wherein the endotoxin-like material is selected from the group consisting of lipopolysaccharide, oligolipopolysaccharide, lipid A, glucans, peptidoglycan, lipoteichoic acid, lipoglycans and synthetic lipid A, the method comprising:
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(a) incubating a sample with a component which is from horseshoe crab amoebocyte lysate, hemolymph or a synthetic analogue thereof having substantially the same reactivity with an endotoxin or endotoxin-like material as a component occurring naturally in horseshoe crab amoebocyte lysate or haemolymph, whereby the properties of said component are altered if any endotoxin or endotoxin-like material is present in the sample so that a reaction product of said component with an endotoxin or endotoxin-like material in the sample will react with an antibody raised against or directed substantially only against said reaction product or an immunological determinant thereof, (b) reacting the incubated mixture of said sample and said component resulting from step (a) with a monoclonal antibody raised against or directed substantially only against said reaction product or an immunological determinant thereof, whereby the antibody is bound to any of said reaction product present in the mixture, and (c) determining the presence of any endotoxin or endotoxin-like material in the sample by detecting any bound antibody in the reaction mixture resulting from step (b), the detection of increasing amounts of bound antibody showing the presence of an endotoxin or endotoxin-like material in the sample; with the proviso that any of said reaction product present after the incubation of the sample in step (a) is coupled to a solid support, or that said antibody is coupled to a solid support or to a bridging molecule which is coupled to a solid support, or that any endotoxin-like material present in the sample is coupled to a solid support. - View Dependent Claims (4)
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Specification