Method of removing lipid inhibitors from limulus amebocyte lysate

• US 4,279,774 A
• Filed: 11/13/1979
• Issued: 07/21/1981
• Est. Priority Date: 11/13/1979
• 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 K.
• Primary Examiner(s): Gron, Teddy S.
• Application Number:
  US06/093,403
• Time in Patent Office: 616 Days
• Field of Search: 23/230 B, 252/408, 424/101, 424/12, 424/95, 210/22 R, 210/21, 435/34, 435/38, 435/259
• US Class Current: 435/18
• CPC Class Codes: G01N 33/579 involving limulus lysate

Abstract

The improved method of the present invention involves the removal of lipid inhibitors from Limulus amebocyte lysate to increase the sensitivity and quality of the lysate. The method comprises intimately contacting, as by mixing and stirring, Limulus amebocyte lysate with a selected binary liquid solvent system for a time sufficient to draw water into the solvent system from the lysate and to effectively extract and denature the lipid inhibitors in the lysate. The ability of the binary solvent to draw in water from the lysate and form, in effect, a new tertiary system is the important factor in this methodology. The amount of water drawn into the solvent is controlled by the amount of polar solvent in the solvent system. After this extraction and denaturation, the lysate is then separated from the solvent system and recovered in purified form of increased sensitivity.

Claims

1. An improved method of removing lipid inhibitors from Limulus amebocyte lysate to increase the sensitivity and quality of the lysate, said method comprising:

(a) intimately contacting Limulus amebocyte lysate with a selected binary liquid solvent system containing first and second selected organic solvents of differing polarities, said first solvent being of low polarity registering not in excess of about 0.5 on the elutotrophic series scale, said second solvent being of high polarity registering at least about 0.8 on said elutotrophic series scale, said second solvent being present in said solvent system in a concentration of about 2-25% by volume of said system, said solvents being present in an amount and contacting said lysate for a time sufficient to draw water into said solvent system from said lysate and extract and denature lipid inhibitors in said lysate; and

,

(b) separating said Limulus amebocyte lysate and said solvent system from each other and recovering said lysate in purified form having improved sensitivity.

2. The improved method of claim 1 wherein said system is used in a volume ratio of about 1:

2 to said lysate.

3. The improved method of claim 1 wherein said first solvent is an aromatic and said second solvent is a low molecular weight alcohol.

4. The improved method of claim 3 wherein said system comprises a major concentration of benzene and a minor concentration of methanol.

5. The improved method of claim 4 wherein said methanol is in a concentration of about 2-5 volume per cent in said system.

6. The improved method of claim 3 wherein said system comprises benzene and ethanol.

7. The improved method of claim 6 wherein said ethanol is present in said system in a concentration of about 2-5 volume percent.

8. The improved method of claim 3 wherein said system comprises toluene and a low molecular weight alcohol.

9. The improved method of claim 8 wherein said alcohol comprises methanol in a concentration of about 2-5 volume percent in said system.

10. The improved method of claim 8 wherein said alcohol comprises ethanol in a concentration of about 2-5 volume percent in said system.

11. The improved method of claim 1 wherein said system comprises a multi-halogenated paraffin and a low molecular weight alcohol.

12. The improved method of claim 11 wherein said system comprises trichloroethane and ethanol, the latter in a concentration of about 2-5 volume percent in said system.

13. The improved method of claim 11 wherein said system comprises trichloroethane and propanol, the latter in a concentration of about 2-5 volume percent in said system.

14. The improved method of claim 1 wherein said contacting is effected by mixing said lysate with said system for approximately one-half hour, and wherein said separating is effected by decanting said system from said lysate, whereupon said lysate is lyophilized.

15. The improved method of claim 1 wherein said first solvent is selected from the group consisting of benzene, trichloroethane and toluene, and mixtures thereof, and wherein said second solvent is selected from the group consisting of methanol, ethanol and propanol, and mixtures thereof.