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

• US 8,440,394 B2
• Filed: 04/11/2011
• Issued: 05/14/2013
• Est. Priority Date: 12/02/2004
• Status: Active Grant

• Current Assignee: National Aeronautics and Space Administration (Government of the United States of America), Charles River Laboratories Incorporated (Pathology Associates, Inc.)
• Original Assignee: Charles River Laboratories Incorporated (Pathology Associates, Inc.)
• Inventors: Wainwright, Norman R., Jordan, Foster T.
• Primary Examiner(s): Afremova, Vera
• Application Number:
  US13/083,996
• Publication Number:
  US 20110256568A1
• Time in Patent Office: 764 Days
• Field of Search: None
• US Class Current: 435/4
• 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 detecting the presence of Gram-positive bacteria in a sample, the method comprising the steps of:

contacting the sample with a crustacean hemocyte preparation comprising a lipoteichoic acid reactive material, wherein the lipoteichoic acid reactive material is activatable by lipoteichoic acid such that prophenol oxidase is converted into phenol oxidase when the lipoteichoic acid reactive material is contacted with lipoteichoic acid; and

detecting the presence or absence of phenol oxidase in the sample, thereby to detect the presence or absence of Gram-positive bacteria in the sample.

2. The method of claim 1, wherein the crustacean hemocyte preparation is produced by the steps of:

(a) providing a crustacean hemocyte preparation, wherein the crustacean 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 crustacean hemocytes in the crustacean hemocyte preparation permeable to the lipoteichoic acid reactive material so that the lipoteichoic acid reactive material is released from the crustacean hemocytes; and

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

3. The method of claim 2, wherein in step (b), the outer membrane is rendered selectively permeable by exposure to a membrane permeabilizing agent.

4. The method of claim 3, wherein the permeabilizing agent is an ionophore.

5. The method of claim 4, wherein the ionophore is a calcium ionophore.

6. The method of claim 4, wherein the ionophore is admixed with the crustacean hemocyte preparation to give a final concentration in the range from about 0.1 μ

M to about 100 μ

M.

7. The method of claim 6, wherein the ionophore is admixed with the crustacean hemocyte preparation to give a final concentration in the range from about 1 μ

M to about 10 μ

M.

8. The method of claim 1, wherein the crustacean is selected from the group consisting of Cancer borealis, Cancer irroratus, Carcinus maenas, Hemigrapsus sanguineus, and Limulus polyphemus.

9. The method of claim 1, wherein the crustacean belongs to the Cancer genus.

10. The method of claim 1, wherein detecting the presence or absence of phenol oxidase in the sample occurs visually.

11. The method of claim 1, wherein detecting the presence or absence of phenol oxidase in the sample is performed by a detector.

12. The method of claim 11, wherein the detector is a spectrophotometer or fluorimeter.

13. The method of claim 1, wherein the sample is contacted with a substrate for phenol oxidase.

14. The method of claim 13, wherein the sample combined with a crustacean hemocyte preparation is contacted with the substrate.

15. The method of claim 13, wherein the presence or absence of phenol oxidase in the sample is detected chromogenically.