Quantitative detection of endotoxin in biological fluids

• US 3,915,805 A
• Filed: 05/29/1973
• Issued: 10/28/1975
• Est. Priority Date: 05/25/1970
• Status: Expired due to Term

• Current Assignee: Jack Levin
• Original Assignee: Jack Levin
• Inventors: Levin, Jack
• Primary Examiner(s): Naff, David M.
• Application Number:
  US05/364,461
• Time in Patent Office: 882 Days
• Field of Search: 195/103.5 23/23B 424/95,101
• US Class Current: 435/4
• CPC Class Codes: G01N 33/579 involving limulus lysate

Abstract

An in vitro method as well as an article is provided for quantitative detection of endotoxin in biologicals, biological fluids, such as blood, and any protein containing material by an indicator comprising amebocyte lysate. This method or technique consists in first centrifuging a blood sample to obtain plasma, and then admixing chloroform with the plasma to precipitate certain protein fractions of the plasma. The mixture of chloroform and plasma is then centrifuged to sediment the precipitated protein and results in the formation of an aqueous layer, an intermediate layer, and a chloroform layer. The intermediate layer is then removed, and a clottable substrate, namely amebocyte lysate is then admixed with the removed intermediate layer. The rate of reaction which is proportional to the concentration of endotoxin in the sample is measured as manifested by the increase in turbidity thereof. The clottable substrate, namely amebocyte lysate, is obtained from amebocytes of Limulus and is provided as an article of manufacture for use in the detection of the endotoxin in the biological fluid sample.

Claims

1. AN IN VITRO METHOD FOR QUANTITATIVE DETECTION OF ENDOTOXIN IN A SAMPLE OF PROTEIN-CONTAINING MATERIAL, COMPRISING:

ADMIXING CHLOROFORM WITH THE SAMPLE TO PRECIPITATE CERTAIN PROTEINS THEREFROM, SETPARATING THE PRECIPITATED PROTEINS FROM THE SAMPLE, ADMIXING LYSTER OF PLASMA-FREE LIMULUS AMEBOCYTES WITH SAID SAMPLE FROM WHICH THE PRECIPITATED PROTEINS HAVE BEEN SEPARATED, AND, MEASURING THE RATE OF REACTION BETWEEN ENDOTOXIN IN THE SAMPLY AND THE LYSATE, THE RATE OF REACTION BEING PROPORTIONAL TO THE CONCENTRATION OF ENDOTOXIN IN THE SAMPLY AS MANIFESTED BY THE INCREASED TURBIDITY OR VISCOSITY THEREOF.

2. The method of claim 1 wherein the second-mentioned step comprises centrifuging the sample from which certain proteins have been precipitated and removing said precipitated proteins from the sample.

3. The method of claim 1 and further comprising:

sedimenting the sample to which chloroform has been added to form an aqueous layer, an intermediate layer, and a chloroform layer in the sample;

removing at least a portion of said intermediate layer; and

, admixing said removed portion of said intermediate layer with lysate of plasma-free Limulus amebocytes.

4. An in vitro method for quantitative detection of endotoxin in a protein containing material, comprising, admixing chloroform with said protein containing material to precipitate certain proteins from said protein containing material, centrifuging said precipitated proteins to obtain a sample contaminated with endotoxin, admixing Limulus amebocyte lysate with said sample, and then measuring the rate of reaction which is proportional to the concentration of endotoxin in said sample as manifested by the increased turbidity or viscosity thereof.

5. An in vitro method for quantitative detection of endotoxin in biological fluids, comprising, admixing chloroform with a biological fluid to precipitate certain proteins of said biological fluid, centrifuging said mixture of chloroform and biological fluid to sediment the precipitated proteins resulting in the formation of an aqueous layer, an intermediate layer, and a chloroform layer, removing a sample from said intermediate layer, admixing Limulus amebocyte lysate with said removed sample, and then measuring the rate of reaction which is proportional to the concentration of endotoxin in said sample as manifested by the increased turbidity or viscosity thereof.

6. An in vitro method for quantitative detection of endotoxin in blood, comprising, admixing chloroform with a blood plasma to precipitate certain proteins of said plasma, centrifuging said mixture of chloroform and plasma to sediment the precipitated proteins resulting in the formation of an aqueous layer, an intermediate layer, and a chloroform layer, removing a sample from said intermediate layer, admixing Limulus amebocyte lysate with said removed sample, and then measUring the rate of reaction which is proportional to the concentration of endotoxin in the sample as manifested by the increased turbidity or viscosity thereof.

7. An in vitro method for quantitative detection of endotoxin in blood, comprising, centrifuging a sample of blood to obtain plasma, admixing chloroform with said plasma to precipitate certain proteins of said plasma, centrifuging said mixture of chloroform and biologial fluid to sediment the precipitated proteins resulting in the formation of an aqueous layer, an intermediate layer, and a chloroform layer, removing a sample from said intermediate layer, admixing Limulus amebocyte lysate with said removed sample layer, and then measuring the rate of reaction which is proportional to the concentration of endotoxin in the sample as manifested by the increased turbidity or viscosity thereof.