Detection of proteases secreted from pathogenic microorganisms
View Patent ↗A diagnostic test kit for detecting the presence or absence of a protease (e.g., aminopeptidase) within a test sample is provided. The test kit comprises a substrate that is capable of being cleaved in the presence of the protease to release a compound. The kit also comprises a lateral flow device that comprises a chromatographic medium. The chromatographic medium defines a detection zone within which is contained a first reagent (e.g., diazonium ion) that is capable of reacting with the compound to form a second reagent (e.g., aromatic azo compound). The second reagent exhibits a color that is different than the color of the first reagent. The lateral flow device also includes an absorbent material located adjacent to the chromatographic medium, the absorbent material receiving the test sample after flowing through the chromatographic medium.
1. A method for detecting the presence of a protease within a test sample, the method comprising:
i) providing a lateral flow assay device that comprises a fluidic medium, the lateral flow assay device comprising a sample application zone, the fluidic medium defining a detection zone within which is immobilized a first reagent, the detection zone being in fluid communication with a substrate, wherein the substrate is capable of cleavage by the protease and a chromogenic product is released upon the cleavage of the substrate by the protease;
ii) contacting the sample application zone with the test sample, wherein the test sample travels through the fluidic medium from the sample application zone to the detection zone following contacting of the sample application zone with the test sample; and
iii) generating a color within the detection zone that corresponds to the presence or absence of the protease in the test sample.
2. The method of claim 1 , further comprising obtaining the test sample from a host, wherein the generated color indicates the presence of a pathogen in the host, wherein the pathogen expresses the protease.
3. The method of claim 2 , wherein the pathogen is a yeast, bacteria, or parasite.
4. The method of claim 2 , wherein the pathogen is pathogenic Candida albicans.
5. The method of claim 2 , wherein the test sample is obtained from saliva.
6. The method of claim 2 , wherein the test sample is obtained from vaginal fluid.
7. The method of claim 1 , further comprising measuring the intensity of the generated color, wherein the amount of the protease in the test sample is proportional to the intensity of the generated color.
8. The method of claim 1 , wherein the protease is a peptidase.
9. The method of claim 8 , wherein the peptidase is an aminopeptidase.
10. The method of claim 8 , wherein the peptidase is proline iminopeptidase.
11. The method of claim 1 , wherein the fluidic medium further defines a second detection zone, the second detection zone being in fluid communication with a second substrate, wherein the second substrate is capable of cleavage by a second protease and a second chromogenic product is released upon the cleavage of the second substrate by the second protease, the method further comprising generating a second color within the second detection zone that corresponds to the presence or absence of the second protease in the test sample.
12. The method of claim 1 , wherein the expression of the protease is upregulated by a hyphal form of Candida albicans.