Reagent for assaying endotoxin

• US 5,605,806 A
• Filed: 04/24/1995
• Issued: 02/25/1997
• Est. Priority Date: 08/22/1990
• Status: Expired due to Term

• Current Assignee: Seikagaku Kogyo Kabushiki Kaisha
• Original Assignee: Seikagaku Kogyo Kabushiki Kaisha
• Inventors: Tanaka, Shigenori, Tamura, Hiroshi
• Primary Examiner(s): Chan, Christina Y.
• Assistant Examiner(s): GRUN, JAMES LESLIE
• Application Number:
  US08/427,260
• Time in Patent Office: 673 Days
• Field of Search: 435/7.32, 435/23, 435/184, 435/962, 436/518, 436/529, 436/530, 436/531, 436/69, 530/413, 530/388.2, 530/388.26, 530/389.1
• US Class Current: 435/7.32
• CPC Class Codes: G01N 33/579 involving limulus lysate
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding

Abstract

The present invention provides a reagent for assaying an endotoxin which comprises limulus amoebocyte lysate and an antibody to (1 → 3)-β-D-glucan sensitive factor or a reagent which comprises a lysate substantially free from (1 → 3)-β-D-glucan sensitive factor. The reagent of the present invention makes it possible to assay an endotoxin originating from gram-negative bacteria contained in a biological sample such as blood, urine and cerebrospinal fluid at an extremely high sensitivity without being affected by (1 → 3)-β-D-glucan.

Claims

1. A reagent for assaying an endotoxin comprising limulus amebocyte lysate and an antibody to (1 →

3)-β

-D-glucan sensitive factor, wherein said antibody specifically binds to and neutralizes (1 →

     3)-β

-D-glucan sensitive factor.

2. The reagent for assaying an endotoxin as claimed in claim 1, wherein said antibody is a monoclonal antibody.

3. The reagent for assaying an endotoxin as claimed in claim 1, wherein said antibody is a polyclonal antibody.

8. The reagent for assaying an endotoxin as claimed in any one of claims 1 to 7, wherein said reagent is a freeze-dried preparation.

4. A reagent for assaying an endotoxin comprising limulus amebocyte lysate, an antibody to (1 →

3)-β

-D-glucan sensitive factor, wherein said antibody specifically binds to and neutralizes (1 →

     3)-β

-D-glucan sensitive factor, and a synthetic peptide substrate for clotting enzyme having an amino acid sequence in common with the hydrolysis sites of coagulogen.

5. The reagent for assaying an endotoxin as claimed in claim 4, wherein said antibody is a monoclonal antibody.

6. The reagent for assaying an endotoxin as claimed in claim 4, wherein said antibody is a polyclonal antibody.

7. A reagent for assaying an endotoxin comprising a limulus amebocyte lysate substantially free from (1 →

3)-β

-D-glucan sensitive factor, obtained by method (I) or (II), wherein said method (I) comprises the steps of;

(a) admixing limulus amebocyte lysate with a carrier onto which an antibody to (1 →

     3)-β

-D-glucan sensitive factor has been immobilized, wherein said antibody specifically binds to and neutralizes (1 →

     3)-β

-D-glucan

sensitive factor, and

(b) removing the carrier from the lysate so as to obtain said reagent;

wherein said method (II) comprises the steps of;

(a) applying limulus amebocyte lysate to a column which has previously been packed with a carrier onto which an antibody to (1 →

     3)-β

-D-glucan sensitive factor has been immobilized, wherein said antibody specifically binds to and neutralizes (1 →

     3)-β

-D-glucan sensitive factor, and

(b) collecting a fraction of the lysate which passes through the column so as to obtain said reagent.

9. The reagent for assaying an endotoxin as claimed in claim 7, wherein said carrier onto which the antibody is immobilized is selected from the group consisting of cellulose, agarose, polyacrylamide, dextran and porous silica beads.

10. A method of assaying for an endotoxin in a sample comprising:

(a) combining the reagent of claim 7 with a sample under conditions whereby endotoxin in the sample, acting via an endotoxin-sensitive factor-dependent cascade, activates clotting enzyme in the reagent,

(b) contacting the activated clotting enzyme with a synthetic peptide substrate for clotting enzyme having an amino acid sequence in common with the hydrolysis sites of coagulogen, and

(c) measuring enzymatic activity of the activated clotting enzyme on coagulogen or on the synthetic peptide substrate as an indication of endotoxin in the sample.

11. A method of assaying for an endotoxin in a sample comprising:

(a) admixing limulus amebocyte lysate with an antibody to (1 →

     3)-β

-D-glucan sensitive factor, wherein said antibody specifically binds to and neutralizes (1 →

     3)-β

-D-glucan sensitive factor in the lysate,

(b) combining the admixed lysate with a sample under conditions whereby endotoxin in the sample, acting via an endotoxin-sensitive factor-dependent cascade, activates clotting enzyme in the lysate,

(c) contacting the activated clotting enzyme with a synthetic peptide substrate for clotting enzyme having an amino acid sequence in common with the hydrolysis sites of coagulogen, and

(d) measuring enzymatic activity of the activated clotting enzyme on coagulogen or on the synthetic peptide substrate as an indication of endotoxin in the sample.