Methods and compositions for the detection of microbial contaminants

• US 7,329,538 B2
• Filed: 03/17/2004
• Issued: 02/12/2008
• 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: Balas, Charles Jr., Wainwright, Norman R., Jordan, Foster T., Rumore, Frank C.
• Primary Examiner(s): Corcoran; Gladys J P
• Assistant Examiner(s): BOWERS, NATHAN ANDREW
• Application Number:
  US10/803,177
• Publication Number:
  US 20040241788A1
• Time in Patent Office: 1,427 Days
• Field of Search: 435/288.7, 435/288.3, 435/288.4, 435/23, 435/287.9
• US Class Current: 435/288.7
• 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 cartridge for determining the presence or amount of a microbial contaminant in a sample, the cartridge comprising:

(i) a housing defining a fluid inlet port, an optical cell, and a conduit having a fluid contacting surface for providing fluid flow communication between the fluid inlet port and the optical cell; and

(ii) a hemocyte lysate and an anti-flaking agent dried on a region of the fluid contacting surface of the conduit, wherein, when a sample is applied to the fluid inlet port, the sample traverses the region and solubilizes the hemocyte lysate during transport to the optical cell.

2. The cartridge of claim 1, further comprising a chromogenic substrate disposed on a second, different region of the fluid contacting surface.

4. The cartridge of claim 2 or 3, wherein the second region is located downstream of the first region.

5. The cartridge of claim 1 or 3, further comprising a preselected amount of an agent representative of the microbial contaminant disposed on the fluid contacting surface of the conduit.

6. The cartridge of claim 5, wherein the agent is disposed on the first region.

7. The cartridge of claim 5, wherein the agent is a bacterial endotoxin or a (1→

3)-β

-D glucan.

15. A method of detecting the presence of a microbial contaminant in a sample, the method comprising the steps of:

(a) introducing a sample into the sample inlet port of the cartridge of claim 1, 8 or 3;

(b) permitting the sample to move to the optical cell; and

(c) measuring an optical property of the sample in the optical cell, wherein a change in the optical property is indicative of the presence of a microbial contaminant in the sample.

16. The method of claim 15, wherein the change in optical property is an increase in absorbance of light of a preselected wavelength.

17. The method of claim 15, wherein the change in optical property is a decrease in transmission of light of a preselected wavelength.

18. A method of determining the amount of a microbial contaminant in a sample, the method comprising the steps of:

(a) introducing a sample into the sample inlet port of the cartridge of claim 1, 8 or 3;

(b) permitting the sample to move to the optical cell;

(c) measuring the time in which a preselected change occurs in an optical property of the sample in the optical cell; and

(d) comparing the time measuring in step (c) against a predetermined standard curve to determine the amount of the microbial contaminant in the sample.

19. The method of claim 18, wherein the change in optical property is an increase in absorbance of light of a preselected wavelength.

20. The method of claim 18, wherein the change in optical property is a decrease in transmission of light of a preselected wavelength.

3. A cartridge for determining the presence or amount of a microbial contaminant in a sample, the cartridge comprising:

(i) a housing defining a fluid inlet port, an optical cell located downstream of the fluid inlet port, a first conduit having a fluid contacting surface for providing fluid flow communication between the fluid inlet port and the optical cell, a pump port located downstream of the optical cell, and a conduit connecting the optical cell and the pump port;

(ii) a hemocyte lysate dried on a first region of the fluid contacting surface of the first conduit; and

(iii) a chromogenic substrate dried on a second, different region of the fluid contacting surface of the first conduit,

wherein, when a sample is applied to the fluid inlet port, the sample traverses the first and second regions and solubilizes the hemocyte lysate and chromogenic substrate during transport to the optical cell.

8. A cartridge for determining the presence or amount of a microbial contaminant in a sample, the cartridge comprising:

(i) a housing defining

a first fluid inlet port, a first optical cell, and a first conduit having a fluid contacting surface for providing fluid flow communication between the first fluid inlet port and the first optical cell, and

a second fluid inlet port, a second optical cell, and a second conduit having a fluid contacting surface for providing fluid flow communication between the second fluid inlet port and the second optical cell;

(ii) a first hemocyte lys ate dried on a first region of the fluid contacting surface of the first conduit, so that when a sample is applied to the first fluid inlet port, the sample traverses the region and solubilizes the first hemocyte lysate during transport to the first optical cell;

(iii) a second hemocyte lysate dried on a first region of the fluid contacting surface of the second conduit, so that when sample is applied to the second fluid inlet port, the sample traverses the region and solubilizes the second hemocyte lysate during transport to the second optical cell, and

(iv) an agent representative of a microbial contaminant dried on the fluid contacting surface of the first conduit.

9. The cartridge of claim 8, further comprising a chromogenic substrate disposed on a second, different region of the fluid contacting surface of the first conduit.

10. The cartridge of claim 9, wherein the second region is located downstream of the first region.

11. The cartridge of claim 9, further comprising a chromogenic substrate disposed on a second, different region of the fluid contacting surface of the second conduit.

12. The cartridge of claim 11, wherein the second region is located downstream of the first region.

13. The cartridge of claim 8, wherein the agent is disposed on the first region.

14. The cartridge of claim 8, wherein the agent is a bacterial endotoxin or a (1→

3)-β

-D glucan.