Methods for the detection of microbial contaminants

• US 7,939,291 B2
• Filed: 08/01/2007
• Issued: 05/10/2011
• Est. Priority Date: 03/17/2003
• Status: Active Grant

• Current Assignee: Charles River Laboratories Incorporated (Pathology Associates, Inc.)
• Original Assignee: Charles River Laboratories Incorporated (Pathology Associates, Inc.)
• Inventors: Jordan, Foster T., Wainwright, Norman R.
• Primary Examiner(s): HOBBS, LISA JOE
• Application Number:
  US11/832,261
• Publication Number:
  US 20080020422A1
• Time in Patent Office: 1,378 Days
• Field of Search: None
• US Class Current: 435/34
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/049 vacuum
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: G01N 2400/24 beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan
• CPC Class Codes: G01N 2400/50 Lipopolysaccharides; LPS
• CPC Class Codes: G01N 33/579 involving limulus lysate

Abstract

The invention provides methods and compositions for the detection and/or quantification of a microbial contaminant, for example, a bacterial endotoxin or a glucan, in a sample. In particular, the invention provides a test cartridge useful in the practice of hemocyte lysate-based assays for the detection and/or quantification of a microbial contaminant in a sample. In addition, the invention provides methods of making and using such cartridges. In addition, the invention provides a rapid, sensitive, multi-step kinetic hemocyte lysate-based assay for the detection and/or quantification of a microbial contaminant in a sample. In addition, the invention provides a glucan-specific lysate that can be used in a variety of assay formats, including, for example, a test cartridge, optionally configured to perform a kinetic assay.

Claims

1. A method of determining the presence of a microbial infection or contamination in a sample, the method comprising the sequential steps of:

(a) contacting a sample with an amebocyte lysate comprising an activatable enzyme to produce a sample-lysate mixture, whereupon the enzyme becomes activated if a microbial contaminant that activates the enzyme is present in the sample;

(b) after step (a), contacting the sample-lysate mixture with a substrate for the enzyme to produce a sample-lysate-substrate mixture, such that, if the mixture contains activated enzyme, the activated enzyme produces a change in the substrate;

(c) determining the time in which a preselected change occurs in an optical property of the sample-lysate-substrate mixture, wherein the change in optical property results from a change in the substrate; and

(d) comparing the time determined in step (c) against a predetermined standard curve to determine whether the microbial infection or contamination is present in the sample.

2. The method of claim 1, wherein the microbial contaminant is selected from the group consisting of a lipopolysaccharide, a bacterial endotoxin, and a glucan.

3. The method of claim 2, wherein the microbial contaminant is a lipopolysaccharide.

4. The method of claim 2, wherein the microbial contaminant is a bacterial endotoxin.

5. The method of claim 2, wherein the microbial contaminant is a glucan.

6. The method of claim 1, wherein the change in optical property is an increase in absorbance of light of a preselected wavelength.

7. The method of claim 1, wherein the change in optical property is a decrease in transmission of light of a preselected wavelength.

8. The method of claim 1, wherein the amebocyte lysate is a Limulus amebocyte lysate.

9. The method of claim 1, wherein the amebocyte lysate is an endotoxin-specific amebocyte lysate.

10. The method of claim 1, wherein the amebocyte lysate is an glucan-specific amebocyte lysate.

11. The method of claim 1, wherein the activatable enzyme is pro-clotting enzyme.

12. The method of claim 1, wherein the activatable enzyme is clotting enzyme.

13. The method in claim 1, wherein the substrate is a chromogenic substrate.

14. The method of claim 13, wherein the chromogenic substrate comprises a para-nitroaniline chromophore.

15. The method of claim 13, wherein the chromogenic substrate comprises Ile-Glu-Ala-Arg-pNA, wherein pNA is a para-nitroaniline group.

16. The method of claim 1, comprising the additional step of measuring the amount of the microbial contaminant in the sample.

17. The method of claim 1, wherein the presence of the microbial contaminant is indicative of a microbial infection or microbial contamination.

18. The method of claim 16, wherein the amount of the microbial contaminant is indicative of a microbial infection or microbial contamination.

19. The method of claim 17 or 18, wherein the microbial infection is a bacterial, yeast, mold, or fungal infection.

20. The method of claim 1, wherein steps (a) and (b) provide a predetermined assay sensitivity and duration.

21. The method of claim 1, wherein steps (a) and (b) are performed in a well defined by a solid support.