Method for detecting endotoxins

• US 4,038,147 A
• Filed: 06/15/1976
• Issued: 07/26/1977
• Est. Priority Date: 06/15/1976
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Reno, Woodrow James
• Primary Examiner(s): Naff, David M.
• Application Number:
  US05/696,429
• Time in Patent Office: 406 Days
• Field of Search: 195/103.5 R, 195/103.5 A, 195/103.5 M, 424/2, 23/230 B
• US Class Current: 435/13
• CPC Class Codes: G01N 33/579 involving limulus lysate
• CPC Class Codes: Y10S 435/947 using protozoa

Abstract

A rapid and reliable method is disclosed for determining the presence or absence of endotoxins in solution, including biological liquids such as transudates and exudates. The method comprises a procedure based upon the alteration of a modified coagulation test system by endotoxins produced by gram-negative microorganisms. The method is particularly useful as reliable aid in the early diagnosis and treatment of infections caused by gram-negative microorganisms and to alert the diagnostician to the possibility of septic shock in an infected host.

Claims

1. A method of determining the presence or absence of endotoxins in liquids, which comprises:

(a) admixing 1 part by volume of mammalian blood plasma, 0.1 to 0.5 parts by volume of mammalian blood platelet factor reagent, 0.1 to 0.4 parts by volume of Limulus polyphemus amebocyte lysate and 0.1 to 0.5 parts by volume of the liquid for determination;

said plasma being first diluted in a ratio of from 1;

4 to 1;

15 (v/v) with an aqueous buffer solution;

(b) incubating the mixture obtained in (a) above at 37°

C. for 10 minutes;

(c) admixing a source of calcium ions with the incubated mixture of (b) above;

(d) incubating the mixture of (c) above at 37°

C. for from 5 to 20 minutes;

(e) disrupting the mixture obtained by incubating in (d) above;

(f) separating solids from the disrupted mixture to obtain a supernatant;

(g) mixing aliquots of the supernatant, a source of calcium ions and mammalian blood plasma;

(h) measuring the elapsed time between completion of mixing in step (g) above and coagulation of the mixture obtained thereby; and

(i) comparing the elapsed time measured in step (h) above with the elapsed time measured after following the steps (a) through (h) above but replacing the liquid for determination added in step (a) with an equal proportion of a known endotoxin free solution.

2. A method according to claim 1 wherein said liquid is the blood serum of a mammal.

3. A method according to claim 1 wherein said plasma is diluted in a ratio of from 1:

4 to 1;

10 (v/v) with an aqueous buffer solution.

4. A method according to claim 1 wherein said separating is by the aid of centrifugal force.

5. A method according to claim 1 wherein said mixing of aliquots in step (g) comprises mixing about 0.1 to 0.5 ml. of a 0.001 to 0.5 molar solution of a calcium salt with 0.1 ml. of said supernatant.

6. A method of determining the presence or absence of endotoxins in blood serum, which comprises:

(a) admixing 1 part by volume of mammalian adsorbed blood plasma with 0.1 to 0.5 parts by volume of mammalian blood platelet factor reagent, 0.1 to 0.4 parts by volume of Limulus polyphemus amebocyte lysate and 0.1 to 0.5 parts by volume of a specimen of blood serum for determination;

said plasma having been first diluted in a ratio of from 1;

4 to 1;

15 (v/v) with an aqueous buffer solution;

(b) incubating the mixture obtained in (a) above at 37°

C. for 10 minutes;

(c) admixing a proportion of a source of calcium ions with the incubated mixture of (b) above;

(d) incubating the mixture of (c) above at 37°

C. for from 15 to 25 minutes;

(e) disrupting the mixture obtained by incubating in (d) above;

(f) separating solids from the disrupted mixture to obtain a supernatant;

(g) mixing aliquots of the supernatant, a source of calcium ions and mammalian blood plasma;

(h) measuring the elapsed time between completion of mixing in step (g) above and coagulation of the mixture obtained thereby; and

(i) comparing the elapsed time with the elapsed time measured following the steps (a) through (h) above but replacing the liquid for determination added in step (a) with an equal proportion of a known endotoxin free blood serum.

7. A method according to claim 6 wherein step (c) comprises adding from 0.1 to 0.5 ml. of 0.001 to 0.5 molar calcium chloride for each ml. of diluted plasma added in step (a).

8. A method according to claim 6 wherein said separating is by the aid of centrifugal force.

9. A method according to claim 6 wherein said mixing of aliquots in step (g) comprises mixing about 0.1 to 0.5 ml. of a 0.001 to 0.5 molar solution of a calcium salt with 0.1 ml. of said supernatant.