Process for measuring endotoxin

• US 5,318,893 A
• Filed: 12/07/1992
• Issued: 06/07/1994
• Est. Priority Date: 02/27/1988
• Status: Expired due to Term

• Current Assignee: WAKO Pure Chemical Industries Limited (Fujifilm Holdings Corporation)
• Original Assignee: WAKO Pure Chemical Industries Limited (Fujifilm Holdings Corporation)
• Inventors: Tsuchiya, Masakazu, Matuura, Shuji
• Primary Examiner(s): Nucker, Christine M.
• Assistant Examiner(s): WOODWARD, MICHAEL P
• Application Number:
  US07/986,300
• Time in Patent Office: 547 Days
• Field of Search: 435/23, 435/18, 435/34, 536/1.1, 536/123
• US Class Current: 435/23
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 2337/00 N-linked chromogens for determinations of peptidases and proteinases
• CPC Class Codes: G01N 2400/24 beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan
• CPC Class Codes: G01N 33/579 involving limulus lysate

Abstract

Measuring of endotoxin using a reaction of a horseshoe crab hemocyte lysate with endotoxin in a solution can be carried out in the presence of a water-soluble polysaccharide containing β-1,3-glucosidic linkage and/or a water-soluble polysaccharide derivative containing β-1,3-glucosidic linkage.

Claims

1. In a process for measuring an amount of endotoxin in a sample containing endotoxin and an unknown amount of interfering contaminants using a reaction of a horseshoe crab hemocyte lysate with said endotoxin, the improvement which comprises performing the reaction in the presence of a curdlan derivative obtained by introducing at least one substituent selected from the group consisting of carboxymethyl, carboxyethyl, methyl, hydroxyethyl, hydroxypropyl and sulfopropyl thereinto.

2. A process according to claim 1, wherein the curdlan derivative is present in the reaction solution in an amount of 100 ng/ml to 100 mg/ml.

3. A process according to claim 1, wherein the water-soluble polysaccharide derivative is present in the reaction solution in an amount of 100 ng/ml to 100 mg/ml.

4. A process for specifically measuring an amount of endotoxin in a sample containing an endotoxin and an unknown amount of interfering contaminants, which comprises mixing a solution of horseshoe crab hemocyte lysate (AL) with said sample in the presence of a curdlan derivative in an amount sufficient to inactivate a factor present in the AL solution which reacts with beta-1,3-glucan, incubating the resulting mixture, analyzing a gel produced and determining the concentration of endotoxin in said sample, said curdlan derivative being obtained by introducing at least one substituent selected from the group consisting of carboxymethyl, carboxyethyl, methyl, hydroxyethyl, hydroxypropyl and sulfopropyl thereinto.

5. A process for specifically measuring an amount of endotoxin in a sample containing an endotoxin and an unknown amount of an interfering contaminants, which comprises mixing a solution of horseshoe crab hemocyte lysate (AL) with said sample in the present of at least one water-soluble polysaccharide derivative selected from the group consisting of derivatives of pachyman, sclerotan, lentinan, schizophyllan, coriolan, laminaran, laminarin, and paramilon, in an amount sufficient to inactivate a factor present in the AL solution which reacts with beta-1,3-glucan, incubating the resulting mixture, analyzing a gel produced and determining the concentration of endotoxin in said sample, said polysaccharide derivative being obtained by introducing at least one substituent selected from the group consisting of carboxymethyl, carboxyethyl, methyl, hydroxyethyl, hydroxypropyl and sulfopropyl thereinto.

6. A process for specifically measuring endotoxin, which comprises mixing a solution of horseshoe crab hemocyte lysate (AL) with a sample in the presence of a curdlan derivative in an amount sufficient to inactivate a factor present in the AL solution which reacts with beta-1,3-glucan, incubating the resulting mixture, and measuring a turbidity due to coagulation, said curdlan derivative being obtained by introducing at least one substituent selected from the group consisting of carboxymethyl, carboxyethyl, methyl, hydroxyethyl, hydroxypropyl and sulfopropyl thereinto.

7. A process for specifically measuring endotoxin, which comprises mixing a solution of horseshoe crab hemocyte lysate (AL) with a sample containing endotoxin in the presence of at least one polysaccharide derivative selected from the group consisting of derivatives of pachyman, sclerotan, lentinan, schizophyllan, coriolan, laminaran, laminarin, and paramilon, in an amount sufficient to inactivate a factor present in the AL solution which reacts with beta-1,3-glucan, incubating the resulting mixture, and measuring a turbidity due to coagulation, said polysaccharide derivative being obtained by introducing at least one substituent selected from the group consisting of carboxymethyl, carboxyethyl, methyl, hydroxyethyl, hydroxypropyl and sulfopropyl thereinto.

8. A process for specifically measuring endotoxin, which comprises mixing a solution of horseshoe crab hemocyte lysate (AL) with a sample containing endotoxin in the presence of a curdlan derivative in an amount sufficient to inactivate a factor present in the AL solution which reacts with beta-1,3-glucan together with a synthetic substrate of protease, incubating the resulting mixture, and measuring a substance released from the synthetic substrate by protease activity, said curdlan derivative being obtained by introducing at least one substituent selected from the group consisting of carboxymethyl, carboxyethyl, methyl, hydroxyethyl, hydroxypropyl and sulfopropyl thereinto.

9. A process for specifically measuring endotoxin, which comprises mixing a solution of horseshoe crab hemocyte lysate (AL) with a sample containing endotoxin in the presence of at least one water-soluble polysaccharide derivative selected from the group consisting of derivatives of pachyman, sclerotan, lentinan, schizophyllan, coriolan, laminaran, laminarin, and paramilon, in an amount sufficient to inactivate a factor present in the AL solution which reacts with beta-1,3-glucan together with a synthetic substrate of protease, incubating the resulting mixture, and measuring a substance released from the synthetic substrate by protease activity, said polysaccharide derivative being obtained by introducing at least one substituent selected from the group consisting of carboxymethyl, carboxyethyl, methyl, hydroxyethyl, hydroxypropyl and sulfopropyl thereinto.

10. A process for specifically measuring endotoxin, which comprises mixing a solution of horseshoe crab hemocyte lysate (AL) with a sample containing endotoxin in the presence of a curdlan derivative in an amount sufficient to inactivate a factor present in the AL solution which reacts with beta-1,3-glucan, incubating the resulting mixture, and measuring a time required for a turbidity change due to coagulation to reach a designated value or a ratio of change in the turbidity, said curdlan derivative being obtained by introducing at least one substituent selected from the group consisting of carboxymethyl, carboxyethyl, methyl, hydroxyethyl, hydroxypropyl and sulfopropyl thereinto.

11. A process for specifically measuring endotoxin, which comprises mixing a solution of horseshoe crab hemocyte lysate (AL) with a sample containing endotoxin in the presence of at least one polysaccharide derivative selected from the group consisting of derivatives of pachyman, sclerotan, lentinan, schizophyllan, coriolan, laminaran, laminarin, and paramilon, in an amount sufficient to inactivate a factor present in the AL solution which reacts with beta-1,3-glucan, incubating the resulting mixture, and measuring a time required for a turbidity change due to coagulation to reach a designated value or a ratio of change in the turbidity, said polysaccharide derivative being obtained by introducing at least one substituent selected from the group consisting of carboxymethyl, carboxyethyl, methyl, hydroxyethyl, hydroxypropyl and sulfopropyl thereinto.

12. In a process for measuring an amount of endotoxin in a sample containing endotoxin and an unknown amount of interfering contaminant using a reaction of a horseshoe crab hemocyte lysate (AL) with said endotoxin, the improvement which comprises preforming the reaction in the presence of an excess amount of at least one water-soluble polysaccharide derivative selected from the group consisting of pachyman, sclerotan, lentinan, schizophyllan, coriolan, laminaran, laminarin, and paramilon, said polysaccharide derivative being obtained by introducing at least one substituent selected from the group consisting of carboxymethyl, carboxyethyl, methyl, hydroxyethyl, hydroxypropyl and sulfopropyl thereinto.