Methods for the detection of microbial contaminants
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.
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.