Reagent for endotoxin assay and method for endotoxin assay using the same

• US 5,695,948 A
• Filed: 06/11/1996
• Issued: 12/09/1997
• Est. Priority Date: 02/26/1993
• Status: Expired due to Term

• Current Assignee: Seikagaku Kogyo Kabushiki Kaisha
• Original Assignee: Seikagaku Kogyo Kabushiki Kaisha
• Inventors: Tanaka, Shigenori, Tamura, Hiroshi, Aita, Kazuhiro
• Primary Examiner(s): LEARY, LOUISE N
• Application Number:
  US08/661,705
• Time in Patent Office: 546 Days
• Field of Search: 435/13, 435/4, 435/18, 435/19, 435/23, 435/24, 435/7.9, 435/29, 435/240.2
• US Class Current: 435/13
• CPC Class Codes: G01N 2333/811 Serine protease (E.C. 3.4.21) inhibitors
• CPC Class Codes: G01N 33/579 involving limulus lysate
• CPC Class Codes: Y10S 435/967 Standards, controls, materials, e.g. validation studies, buffer systems

Abstract

This invention provides (1) a reagent for endotoxin assay which comprises aprotinin and a limulus amebocyte lysate reagent, (2) a kit for endotoxin assay which comprises the limulus amebocyte lysate reagent and a reagent containing aprotinin, (3) a method for assaying endotoxin in a sample using the limulus amebocyte lysate reagent in which aprotinin is added to the lysate reagent and/or the sample, (4) a method for assaying endotoxin in a serine protease-containing sample using the limulus amebocyte lysate reagent in which the sample is allowed to contact with an aprotinin-immobilized insoluble carrier in advance of endotoxin assay, (5) a carrier for pretreating a serine protease-containing sample on which aprotinin is immobilized, (6) a method for inhibiting factor G activation in which aprotinin is added to the limulus amebocyte lysate reagent and (7) a factor G activation inhibitor which comprises aprotinin as an active ingredient.

Endotoxin assay can be effected based on the factor C system reaction, without influences of factor G contained in the limulus amebocyte lysate reagent and/or serine proteases contained in samples.

Claims

1. A method for specifically assaying for endotoxin in a sample comprising the steps of:

(A) adding aprotinin to a horseshoe crab amebocyte lysate reagent,

(B) adding the resulting lysate reagent of step (A) to the sample, and

(C) assaying for endotoxin in the resulting mixture of Step (B),

wherein said aprotinin is added to the lysate reagent in Step (A) in an amount sufficient to completely inhibit activation of factor G present in the horseshoe crab amebocyte lysate reagent.

2. The method according to claim 1, wherein assaying for endotoxin in Step (C) is carried out by determining the activity of clotting enzyme.

3. A method for specifically assaying for endotoxin in a sample comprising the steps of:

(A) adding aprotinin to the sample,

(B) adding a horseshoe crab amebocyte lysate reagent to the resulting sample of Step (A), and

(C) assaying for endotoxin in the resulting mixture of Step (B),

wherein said aprotinin is added to the sample in Step (A) in an amount sufficient to completely inhibit activation of factor G present in the horseshoe crab amebocyte lysate reagent.

4. The method according to claim 3, wherein assaying for endotoxin in Step (C) is carried out by determining the activity of clotting enzyme.

5. A method for specifically assaying for endotoxin in a sample comprising the steps of:

(A) adding aprotinin to a horseshoe crab amebocyte lysate reagent,

(B) adding aprotinin to the sample,

(C) combining the resulting lysate reagent of step (A) and the resulting sample of step (B) to form a mixture, and

(D) assaying for endotoxin in the mixture of step (C),

wherein said aprotinin is added to the lysate reagent of Step (A) and to the sample of Step (B) in an amount sufficient to completely inhibit activation of factor G present in the horseshoe crab amebocyte lysate reagent.

6. The method according to claim 5, wherein assaying for endotoxin in Step (D) is carried out by determining the activity of clotting enzyme.

7. A method for inhibiting activation of factor G present in a horseshoe crab amebocyte lysate reagent, comprising the step of adding aprotinin to said lysate reagent, wherein said aprotinin is added in an amount sufficient to completely inhibit activation of factor G in said reagent.