Methods and compositions for the detection and/or quantification of gram positive bacterial contaminants

• US 20060183181A1
• Filed: 12/01/2005
• Published: 08/17/2006
• Est. Priority Date: 12/02/2004
• Status: Active Grant

• Current Assignee: Charles River Laboratories Incorporated (Pathology Associates, Inc.)
• Original Assignee: Charles River Laboratories Incorporated (Pathology Associates, Inc.)
• Inventors: Wainwright, Norman R., Jordan, Foster T.
• Granted Patent: US 7,968,280 B2
• Time in Patent Office: Days
• US Class Current: 435/25
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: G01N 33/579 involving limulus lysate
• CPC Class Codes: G01N 33/80 involving blood groups or blood types or red blood cells white blood cells G01N33/56972
• CPC Class Codes: G01N 33/92 involving lipids, e.g. cholesterol , lipoproteins, or their receptors steroid hormones G01N33/743

Abstract

The invention provides methods and compositions for the detection and/or quantification of a Gram positive bacterial contaminant in a sample. In particular, the invention provides hemocyte-based preparations, methods of making such hemocyte-based preparations, and methods of using such hemocyte-based preparations for the detection and/or quantification of the Gram positive bacterial contaminant.

Claims

1. A method of producing a preparation for detecting the presence and/or amount of lipoteichoic acid in a sample, the method comprising the steps of:

(a) providing a hemocyte preparation harvested from a crustacean selected from the group consisting of Cancer borealis, Cancer irroratus, Hemigrapsus sanguineus, and Limulus polyphemus, wherein the hemocytes contain lipoteichoic acid reactive material disposed therein;

(b) releasing the lipoteichoic acid reactive material from the hemocytes; and

(c) harvesting the lipoteichoic reactive material released from the hemocytes.

2. The method of claim 1, wherein in step (b) the lipoteichoic acid reactive material is released by lysis of the hemocytes.

3. The method of claim 1, wherein in step (b) the lipoteichoic acid reactive material is released when the hemocytes are rendered permeable to lipoteichoic acid reactive.

4. The method of claim 3, wherein the hemocytes are rendered permeable by exposure to a membrane permeabilizing agent.

5. The method of claim 4, wherein the permeabilizing agent is an ionophore.

6. The method of claim 5, wherein the ionophore is a calcium ionophore.

7. The method of claim 5, wherein the ionophore is admixed with the hemocyte preparation to give a final concentration in the range from about 0.1 μ

M to about 100 μ

M.

8. The method of claim 7, wherein the ionophore is admixed with the hemocyte preparation to give a final concentration in the range from about 1 μ

M to about 10 μ

M.

9. The method of claim 5, wherein the membrane is rendered permeable by exposing the membrane to the ionophore in the presence of divalent cations.

10. The method of claim 9, wherein the cations are calcium ions.

11. The method of claim 1, wherein the crustacean is Limulus polyphemus.

12. The method of claim 1, wherein the lipoteichoic acid reactive material comprises pro-phenol oxidase.

13. The method of claim 1, wherein the lipoteichoic acid reactive material comprises an enzyme, which is not activated when released from the hemocytes but when later activated converts pro-phenol oxidase into phenol oxidase.

14. The method of claim 1, comprising an additional step of, after step (c), combining the harvested lipoteichoic reactive material with a substrate for phenol oxidase.

56. A preparation of lipoteichoic acid reactive material produced by the method of claim 1.

70. A method of detecting the presence of Gram-positive bacteria in a sample, the method comprising contacting the sample with the preparation of claim 56.

15. A method of producing a preparation for detecting the presence and/or quantity of lipoteichoic acid in a sample, the method comprising the steps of:

(a) providing a hemocyte preparation, wherein hemocytes in the preparation (i) have an outer membrane and (ii) contain lipoteichoic acid reactive material disposed therein;

(b) rendering the outer membrane of at least a portion of the hemocytes in the hemocyte preparation permeable to the lipotechoic acid reactive material so that the lipoteichoic acid reactive material is released from the hemocytes; and

(c) harvesting the lipoteichoic acid reactive material released from the hemocytes.

16. The method of claim 15, wherein in step (b), the outer membrane is rendered selectively permeable by exposure to a membrane permeabilizing agent.

17. The method of claim 16, wherein the permeabilizing agent is an ionophore.

18. The method of claim 17, wherein the ionophore is a calcium ionophore.

23. The method of claim 15, wherein the hemocytes are derived from a crustacean.

24. The method of claim 23, wherein the crustacean is selected from the group consisting of Cancer borealis, Cancer irroratus, Carcinus maenas, Hemigrapsus sanguineus, and Limulus polyphemus.

25. The method of claim 23, wherein the crustacean is Cancer borealis or Limulus polyphemus.

26. The method of claim 15, wherein the lipoteichoic acid reactive material comprises pro-phenol oxidase.

27. The method of claim 15, wherein the lipoteichoic acid reactive material comprises an enzyme, which when activated converts pro-phenol oxidase into phenol oxidase.

28. The method of claim 15 comprising the additional step of, after step (c), combining the harvested lipoteichoic acid reactive material with a substrate for phenol oxidase.

19-22. -22. (canceled)

29. A method of producing a preparation of lipoteichoic acid reactive material, the method comprising the steps of:

(a) providing a hemocyte preparation, wherein hemocytes in the preparation (i) have an outer membrane and (ii) contain lipoteichoic acid reactive material disposed therein;

(b) admixing the hemocytes with a membrane permeabilizing agent to release the lipoteichoic acid reactive material from at least a portion of the hemocytes; and

(c) harvesting the lipoteichoic acid reactive material released from the hemocytes.

30. The method of claim 29, wherein the permeabilizing agent makes the outer membrane selectively permeable to the lipoteichoic acid reactive material.

31. The method of claim 30, wherein the permeabilizing agent is an ionophore.

32. The method of claim 31, wherein the ionophore is a calcium ionophore.

37. The method of claim 29, wherein the hemocytes are derived from a crustacean.

40. The method of claim 29, wherein the lipoteichoic acid reactive material comprises pro-phenol oxidase.

41. The method of claim 29, wherein the lipoteichoic acid reactive material comprises an enzyme, which when activated converts pro-phenol oxidase into phenol oxidase.

42. The method of claim 29 comprising the additional step of, after step (c), combining the harvested lipoteichoic acid reactive material with a substrate for phenol oxidase.

33-36. -36. (canceled)

38-39. -39. (canceled)

43-55. -55. (canceled)

57-59. -59. (canceled)

60. A composition comprising an isolated enzyme preparation derived from crustacean hemocytes, wherein the enzyme preparation comprises pro-phenol oxidase which is converted into phenol oxidase when the enzyme preparation is contacted with lipoteichoic acid.

61. The composition of claim 60, wherein the enzyme preparation is derived from hemocytes whose outer membranes have been rendered selectively permeable to pro-phenol oxidase.

63. The composition of claim 60, wherein the crustacean is Cancer borealis.

64. The composition of claim 60, wherein the enzyme preparation is substantially free of Factor C activity, Factor G activity, or both Factor C and Factor G activity.

65. The composition of claim 60, wherein the enzyme preparation remains substantially inactivated in the presence of (1→

3)-β

-D glucan, lipopolysaccharide, or proteoglycan.

69. The composition of claim 60 further comprising a substrate for phenol oxidase.

73. A method of detecting the presence of Gram-positive bacteria in a sample, the method comprising contacting the sample with the composition of claim 60.

62. (canceled)

66. (canceled)

67. A crustacean hemocyte lysate comprising pro-phenol oxidase, which is converted into phenol oxidase when the hemocyte lysate is contacted with lipoteichoic acid.

68. (canceled)

71-72. -72. (canceled)

74-75. -75. (canceled)