Chromogenic method of detecting endotoxins in blood

• US 4,301,245 A
• Filed: 05/29/1980
• Issued: 11/17/1981
• Est. Priority Date: 05/29/1980
• Status: Expired due to Term

• Current Assignee: Biowhittaker Technologies, Inc. (Lonza Group AG)
• Original Assignee: Dynasciences Corp. (Meggitt PLC)
• Inventors: O'Beirne, Andrew J., Lindsay, Gene
• Primary Examiner(s): Castel, Benoit
• Application Number:
  US06/154,360
• Time in Patent Office: 537 Days
• Field of Search: 435/4, 435/13, 435/23, 435/24, 435/7, 435/259, 435/188, 435/19, 23/230 B, 424/95, 424/101, 252/408 R
• US Class Current: 435/4
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/579 involving limulus lysate
• CPC Class Codes: Y10S 930/28 Bound to a nonpeptide drug, nonpeptide label, nonpeptide carrier, or a nonpeptide resin

Abstract

An improved method is provided for detecting endotoxins in blood serum and/or plasma, particularly human blood fractions. The method employs king crab amebocyte lysate, preferably Limulus amebocyte lysate, in the presence of a substrate which has a selected colorimetric indicator bound to it. The indicator is capable of being split from the substrate by an enzyme which can be generated in the lysate by endotoxins in the blood. Thus, the endotoxins convert proenzyme in the lysate to the enzyme which effects the splitting off of the colorimetric indicator from the substrate. The endotoxin concentration in the blood can thus be determined colorimetrically, that concentration being proportional to the concentration of color indicator split from the substrate. The blood sample need not be extracted, as is required in prior methods, with a solvent such as chloroform to remove inhibitors therein which would interfere with a lysate gelation reaction. Heparin is utilized in the present method in a concentration sufficient to stabilize the lysate against loss of potency but insufficient to inhibit the reaction whereby endotoxin generates the described splitting enzyme from proenzyme in the lysate and the reaction of the enzyme to cause the split. The blood fraction need not be diluted with water and the lysate may be one which has been reconstituted from dry powder, if desired. The method is simple, highly effective, reproducible, inexpensive and rapid.

Claims

1. An improved chromogenic method of detecting endotoxins in blood, which method comprises:

a. reacting an untreated blood fraction sample which comprises serum and/or plasma with king crab amebocyte lysate and a selected substrate containing a selected colorimetric indicator capable of being split from said substrate by an enzyme, said reaction being carried out for a time sufficient to cause any endotoxin present in the blood fraction to effect the generation in the lysate of enzyme capable of splitting off said colorimetric indicator from said substrate and to cause said splitting to occur; and

,

b. thereafter determining said endotoxin concentration colorimetrically, said endotoxin concentration being proportional to the concentration of said color indicator split from said substrate.

2. The improved chromogenic method of claim 1 wherein said endotoxin converts proenzyme in said lysate to said enzyme and wherein said blood is human blood.

3. The improved chromogenic method of claim 2 wherein said blood sample contains a sufficient concentration of blood inhibitors to the gelation reaction which normally occurs between said lysate and endotoxin which may be present in said blood sample so as to substantially inhibit said gelation reaction.

4. The improved chromogenic method of claim 2 wherein said blood sample is essentially undiluted with water.

5. The improved chromogenic method of claim 2 wherein said lysate is Limulus amebocyte lysate and contains an amount of heparin sufficient to stabilize said lysate against loss of potency but insufficient to inhibit said endotoxin-lysate reaction, said amount of heparin being present in a concentration of at least about 0.1 and less than 0.8 units per milliliter of said lysate.

6. The improved chromogenic method of claim 5 wherein said heparin concentration is about 0.1-0.4 unit per milliliter of said lysate.

7. The improved chromogenic method of claim 5 wherein said lysate has been reconstituted from dry powder.

8. The improved chromogenic method of claim 7 wherein said heparin has been added to said lysate after said lysate has been reconstituted.

9. The improved chromogenic method of claim 5 wherein said heparin is in the form of an ammonium salt thereof.

10. A method of stabilizing king crab amebocyte lysate against loss of potency, said method comprising combining with said lysate an amount of heparin sufficient to stabilize said lysate against loss of potency but insufficient to inhibit the reaction between said lysate and endotoxins, said amount of heparin being present in a concentration of at least about 0.1 and less than 0.8 units per milliliter of said lysate.

11. The method of claim 10 wherein said heparin concentration is about 0.1-0.4 units per milliliter of said lysate.