Process for measuring amount of endotoxin or (1.fwdarw.3)-.beta.-D-glucan by kinetic turbidimetric method

• US 5,637,474 A
• Filed: 10/19/1995
• Issued: 06/10/1997
• Est. Priority Date: 10/31/1994
• Status: Expired due to Fees

• Current Assignee: WAKO Pure Chemical Industries Limited (Fujifilm Holdings Corporation)
• Original Assignee: WAKO Pure Chemical Industries Limited (Fujifilm Holdings Corporation)
• Inventors: Kawabe, Keiko, Takaoka, Aya, Ijiri, Haruhisa, Tsuchiya, Masakazu
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US08/545,290
• Time in Patent Office: 600 Days
• Field of Search: 435/4, 435/18, 435/23, 435/34
• US Class Current: 435/18
• CPC Class Codes: G01N 33/579 involving limulus lysate

Abstract

A process for measuring the amount of endotoxin or (1→3)-β-D-glucan (referred to hereinafter as β-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 β-glucan contained in the sample on the basis of the relationship between the said time and the amount of endotoxin or β-glucan; and also a reagent for measuring the amount of endotoxin or β-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.

Claims

1. A process for determining an amount of endotoxin or of (1→

3)-β

-D-glucan contained in a sample, which comprises the steps of;

mixing the sample with horseshoe crab amebocyte lysate, in the absence of an ionic surfactant, with at least one water-soluble polymer selected from the group consisting of polyethylene glycol, polyvinyl alcohol, methyl cellulose and hydroxypropyl cellulose, to form a liquid mixture;

applying a light to the liquid mixture;

measuring a time required until a predetermined degree of change in transmittance, in absorbance, or in R_t or log R_t, wherein R_t =I_t /I₀, and wherein I₀ is the amount of initially transmitted light and I_t is the amount of light transmitted after time t elapsed from the mixing of the sample with the amebocyte lysate, or from a predetermined time from the mixing of the sample with the amebocyte lysate; and

determining the amount of the endotoxin or of the (1→

3)-β

-D-glucan contained in the sample by correlating the time measured in the preceding step with a previously prepared calibration curve using the time required for the liquid mixture to reach the aforesaid predetermined degree of change after the mixing of the sample with the amebocyte lysate, or after a predetermined time from the mixing of the sample with the amebocyte lysate.

2. A process according to claim 1, wherein the water-soluble polymer is a polyethylene glycol.

3. A process according to claim 2, wherein the polyethylene glycol has a weight-average molecular weight of 3,000 to 4,000,000.

4. A reagent for measuring an amount of endotoxin or of (1→

3)-β

-D-glucan contained in a sample, said reagent consisting essentially of horseshoe crab amebocyte lysate and at least one water-soluble polymer selected from the group consisting of polyethylene glycol, polyvinyl alcohol, methyl cellulose and hydroxypropyl cellulose.