Process for measuring endotoxin

• US 5,179,006 A
• Filed: 02/23/1989
• Issued: 01/12/1993
• 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:
  US07/313,829
• Time in Patent Office: 1,419 Days
• Field of Search: 435/23, 435/18, 435/34
• 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 an unknown amount of endotoxin by using a reaction of a horseshoe crab hemocyte lysate with endotoxin in a solution, the improvement which comprises carrying out the reaction in the presence of at least one water-soluble polysaccharide selected from the group consisting of curdlan, pachyman, scleratan, lentinan, schizophyllan, coriolan, laminaran, laminarin, paramilon and carboxyne thylated curdlan, in an mount sufficient to inactivate a factor present in the AL solution which reacts with beta-1,3-glucan, and determining the concentration of endotoxin in said unknown sample.

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

7. The process of claim 1, wherein said polysaccharide derivative is carboxymethyl curdlan.

3. A process for specifically measuring an amount of endotoxin in a sample containing an unknown amount of endotoxin which comprises mixing a solution of horseshoe crab hemocyte lysate with said sample in the presence of at least one water-soluble polysaccharide selected from the group consisting of curdlan, pachyman, scleratan, lentinan, schizophyllan, coriolan, laminaran, laminarin, paramilon and carbosymethylated curdlan, in an amount sufficient to inactive ate a factor present in the AL solution which reacts with beta-1,3-glucan, incubating the resulting mixture, judging a gel produced and determining the concentration of endotoxin in said sample.

8. The process of claim 3, wherein said polysaccharide derivatives is carboxymethyl curdlan.

4. A process for specifically measuring endotoxin, which comprises mixing a solution of horseshoe crab hemocyte lysate with a sample in the presence of at least one polysaccharide selected from the group consisting of curdlan, pachyman, scleratan, lentinan, schizophyllan, coriolan, laminaran, laminarin, paramilon and derivatives thereof, 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.

9. The process of claim 4, wherein said polysaccharide derivative is carboxymethyl curdlan.

5. A process for specifically measuring endotoxin, which comprises mixing a solution of horseshoe crab hemocyte lysate with a sample int eh presence of at least one water-soluble polysaccharide selected from the group consisting of curdlan, pachyman, scleratan, lentinan, schizophyllan, coriolan, laminaran, laminarin, paramilon and carboxcymethylated cardlan, 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.

6. A process for specifically measuring endotoxin, which comprises mixing a solution of horseshoe crab hemocyte lysate with a sample in the presence of at least one water-soluble polysaccharide selected from the group consisting of curdlan, pachyman, scleratan, lentinan, schizophyllan, coriolan, laminaran, laminarin, paramilon and derivatives thereof, in an amount sufficient to inactivate a factor present in the AL solution which reacts with beta-1,3-glucan, incubating the resulting mixture, ad measuring a time required for a turbidity change due to coagulation to reach a designed value or a ratio of change in the turbidity.

10. The process of claim 6, wherein said polysaccharide derivatives are obtained by introducing at least one of a carboxymethyl group, a carboxyethyl group, a methyl group, a hydroxyethyl group, a hydroxypropyl group and a sulfopropyl group into curdlan, pachyman, scleratan, lentinan, chizophyllan, coriolan, laminaran, laminarin or paramilon.

11. The process of claim 6, wherein said polysaccharide derivative is carbvoxymethyl curdlan.