Detection of degradative enzymes in bodily fluids
Provided herein are methods useful in detecting degradative enzymes in bodily fluid samples.
1. A method of detecting a degradative enzyme in a bodily fluid sample, said method comprising the steps of:
(i) contacting said bodily fluid sample with a negatively charged degradative enzyme substrate or neutral degradative enzyme substrate;
(ii) allowing said degradative enzyme to react with said negatively charged degradative enzyme substrate or neutral degradative enzyme substrate thereby forming a positively charged degradative enzyme product;
(iii) electrophoretically separating said positively charged degradative enzyme product from said negatively charged degradative enzyme substrate or neutral degradative enzyme substrate; and
(iv) detecting said separated positively charged degradative enzyme product thereby detecting said degradative enzyme in said bodily fluid sample.
2. The method of claim 1 , wherein said negatively charged degradative enzyme substrate or neutral degradative enzyme substrate and said positively charged degradative enzyme product comprise a detectable moiety.
3. The method of claim 2 , wherein said detectable moiety is a fluorophore, a quantum dot, a fluorescent nanoparticle, a dendrimeric nanoparticle, a metallic nanoparticle, a chemiluminescent label, a electrochemical label or an oxidation/reduction label.
4. The method of claim 2 , wherein said detectable moiety is a fluorophore.
5. The method of claim 4 , wherein said detecting in step (iv) comprises detecting said fluorophore.
6. The method of claim 2 , wherein said detectable moiety is a chemiluminescent label.
7. The method of claim 6 , wherein said detecting in step (iv) comprises detecting said chemiluminescent label.
8. The method of claim 1 , wherein said positively charged degradative enzyme product in step (iii) is separated from detectable compounds of said bodily fluid sample.
9. The method of claim 8 , wherein said detectable compounds of said bodily fluid sample are fluorescent.
10. The method of claim 1 , wherein said bodily fluid sample is a crude bodily fluid sample.
11. The method of claim 1 , wherein said bodily fluid sample is a crude blood sample or a crude lymphoid fluid sample.
12. The method of claim 1 , wherein said bodily fluid sample is a crude blood sample.
13. The method of claim 1 , wherein said bodily fluid sample is a blood plasma sample.
14. The method of claim 1 , wherein said negatively charged degradative enzyme substrate or neutral degradative enzyme substrate is a peptide, a lipid, a polysaccharide or a nucleic acid.
15. The method of claim 1 , wherein said negatively charged degradative enzyme substrate or neutral degradative enzyme substrate is a peptide.
16. The method of claim 1 , wherein said degradative enzyme is a protease, a lipase, an amylase or a nuclease.
17. The method of claim 1 , wherein said degradative enzyme is a protease.
18. The method of claim 17 , wherein said protease is a serine protease, a threonine protease, a cysteine protease, an aspartic acid protease, a metalloprotease or a glutamic acid protease.
19. The method of claim 1 , wherein said process of electrophoretically separating is performed using a gel electrophoresis.
20. The method of claim 19 , wherein said gel electrophoresis is a gradient gel electrophoresis.
21. The method of claim 10 , wherein about 100 to about 1000 μl of said crude bodily fluid sample are contacted with said negatively charged degradative enzyme substrate or neutral degradative enzyme substrate.
22. The method of claim 10 , wherein about 1 to about 100 μl of said crude bodily fluid sample are contacted with said negatively charged degradative enzyme substrate or neutral degradative enzyme substrate.
23. The method of claim 10 , wherein about 0.1 to about 10 μl of said crude bodily fluid sample are contacted with said negatively charged degradative enzyme substrate or neutral degradative enzyme substrate.
24. The method of claim 1 , wherein said negatively charged degradative enzyme substrate or neutral degradative enzyme substrate is selected from the group consisting of the following peptides: SEQ ID 1 to 18 and conservative amino acid substitutions thereof.
25. The method of claim 1 , wherein the differential in charge between said negatively charged degradative enzyme substrate or neutral degradative enzyme substrate and said positively charged degradative product results in a 10 to 100 fold increase in detection sensitivity for said positively charged degradative product.