Methods and compositions for the detection of bacterial endotoxins

• US 6,270,982 B1
• Filed: 10/09/1997
• Issued: 08/07/2001
• Est. Priority Date: 10/09/1997
• Status: Expired due to Term

• Current Assignee: Crl Holdings, Inc. (Bausch Health Cos., Inc.)
• Original Assignee: Charles River Laboratories Incorporated (Pathology Associates, Inc.)
• Inventors: Wainwright, Norman R., Jordan, Foster T., Cooper, James F., Chiang, Hui-Ti
• Primary Examiner(s): Swartz, Rodney P
• Application Number:
  US08/947,584
• Time in Patent Office: 1,398 Days
• Field of Search: 424/284.1, 424/184.1, 424/234.1, 424/278.1, 424/282.1, 435/34, 435/184, 435/962, 435/4, 435/702, 435/7.32, 514/23
• 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 invention provides methods and compositions for the detection and/or quantification of bacterial endotoxins. In particular, provided herein is an inexpensive and reproducible method for producing an improved amebocyte lysate preparation having reduced Factor G activity. Provided also is an endotoxin-specific amebocyte lysate preparation produced by such a method. In addition, the invention provides methods and compositions for enhancing the sensitivity to endotoxins of amebocyte lysate preparations having reducing Factor G activity. In particular, the sensitivity of such amebocyte lysate preparations to endotoxins can be enhanced by the addition of exogenous (1→3) β-D-glucan.

Claims

1. A method of producing an improved amebocyte lysate preparation having reduced Factor G activity, the method comprising the steps of:

(a) admixing crude amebocyte lysate with an ionic surfactant in an amount sufficient to produce a solution containing a precipitate; and

(b) separating said precipitate from said solution thereby to produce an amebocyte lysate preparation which is less reactive with (1→

3)-β

-D glucan than said crude amebocyte lysate.

2. The method of claim 1, wherein said amebocyte lysate preparation is reactive with a bacterial endotoxin.

3. The method of claim 1 or 2, wherein said precipitate contains Factor G.

4. The method of claim 1 or 2, wherein said ionic surfactant is a zwitterionic surfactant.

5. The method of claim 4, wherein said zwitterionic surfactant is a sulfobetaine-type surfactant.

6. The method of claim 5, wherein said sulfobetaine-type surfactant is selected from the group consisting of n-octyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, and n-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate.

7. The method of claim 5, wherein said sulfobetaine-type surfactant is n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate.

8. The method of claim 1, comprising the additional step of removing said ionic surfactant from said solution.

9. The method of claim 8, wherein said ionic surfactant is removed by organic solvent extraction.

10. The method of claim 9, wherein said organic solvent is chloroform.

11. A method of producing an improved amebocyte lysate preparation having reduced Factor G activity, the method comprising the steps of:

(a) admixing crude amebocyte lysate with a zwitterionic surfactant in an amount sufficient to produce a solution containing a precipitate; and

(b) removing said precipitate from said solution thereby to produce an amebocyte lysate preparation which is less reactive with (1→

3)-β

-D glucan than said crude amebocyte lysate.

12. The method of claim 11, wherein said amebocyte lysate preparation reacts with a bacterial endotoxin.

13. The method of claim 11, wherein said precipitate contains Factor G.

14. The method of claim 11, wherein said zwitterionic surfactant is a sulfobetaine-type surfactant.

15. The method of claim 14, wherein said sulfobetaine-type surfactant is selected from the group consisting of n-octyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, and n-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate.

16. The method of claim 14, wherein said sulfobetaine surfactant is n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate.

17. The method of claim 11, comprising the additional step of removing said zwitterionic surfactant from said solution.

18. The method of claim 17, wherein said zwitterionic surfactant is removed by organic solvent extraction.

19. The method of claim 18, wherein said organic solvent is chloroform.

23. The method of claim 2, 12, or 20 comprising the additional step of, after step (b) of claim 2 or 12 or after step (c) of claim 20, adding exogenous (1→

3)-β

-D glucan to said amebocyte lysate preparation in an amount sufficient to enhance the sensitivity of said amebocyte lysate preparation to said endotoxin relative to said amebocyte lysate preparation without said exogenous (1→

3)-β

-D glucan.

24. The method of claim 23, wherein said exogenous (1→

3)-β

-D glucan is selected from the group consisting of LAL-RM, curdlan, pachyman, scleratan, leutinan, schizophyllan, coriolan, laminaran, and laminarin.

25. The method of claim 23, wherein said (1→

3)-β

-D glucan is laminarin.

26. An amebocyte lysate preparation produced by the method of claim 23.

27. In an amebocyte lysate-based method of detecting a bacterial endotoxin in a sample, the improvement comprising using the amebocyte lysate preparation of claim 26.

20. A method of producing an improved amebocyte lysate having reduced Factor G activity, the method comprising the steps of:

(a) admixing crude amebocyte lysate with a n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate surfactant in an amount sufficient to produce a solution containing a precipitate;

(b) separating said precipitate from said solution; and

(c) removing said n-tetradecyl-N, N-dimethvl-3-ammonio-1-propanesulfonate surfactant from said solution thereby to produce an amebocyte lysate preparation that is reactive with bacterial endotoxin and is less reactive with (1→

3)-β

-D glucan than said crude amebocyte lysate.

21. The method of claim 20, wherein said n-tetradecyl-N N-dimethyl-3-ammonio-1-propanesulfonate surfactant is removed by organic solvent extraction.

22. The method of claim 21, wherein said organic solvent is chloroform.