Metalloprotease assay
View Patent ↗An assay to detect a metalloprotease in a sample, comprising contacting the sample with a substrate. The metalloprotease reacts with the substrate to form a product comprising a tag. This is followed by selectively binding the tag to a solid phase, wherein the solid phase comprises a binding partner for the tag. Measuring the mass of the product takes place to determine the presence of the metalloprotease in the sample.
1. An assay to detect a metalloprotease in a sample, comprising:
contacting the sample with a substrate, wherein the metalloprotease reacts with the substrate to form a product comprising a tag; followed by
selectively binding the tag to a solid phase, wherein the solid phase comprises a binding partner for the tag; and
measuring the mass of the product, to determine the presence of the metalloprotease in the sample.
2. An assay according to claim 1 wherein the metalloprotease cleaves the substrate to form a product having a lower mass than the substrate.
3. An assay according to claim 1 wherein
the assay is for detecting whether either or both of a first metalloprotease and a second metalloprotease are present in the sample,
the first metalloprotease cleaves the substrate to form a first product having a first mass and comprising a first tag, and the second metalloprotease cleaves the substrate to form a second product having a second mass and comprising a second tag,
wherein the mass of the first product is different from the mass of the second product, and selectively binding the first tag to a solid phase, wherein the solid phase comprises a binding partner for the first tag, and selectively binding the second tag to a solid phase, wherein the solid phase comprises a binding partner for the second tag, and
measuring the respective masses of the products to determine the presence of the first product, the second product or both the first product and the second product.
4. An assay according to claim 1 wherein the tag comprises a peptide sequence which binds to a hydrophobic surface.
5. An assay according to claim 1 wherein the tag binds to an antibody.
6. An assay according to claim 1 wherein the tag binds to biotin or streptavidin.
7. An assay according to claim 1 wherein the binding partner is specific for the tag.
8. An assay according to claim 7 wherein the binding partner is an antibody specific for the tag.
9. An assay according to claim 1 , wherein the substrate is cleaved by botulinum toxin, anthrax toxin or anthrax lethal factor.
10. An assay according to claim 1 , wherein the substrate comprises:
(a) VAMP,
(b) SNAP-25,
(c) syntaxin,
(d) a fragment of any of (a)-(c) comprising a metalloprotease cleavage site, or
(e) a metalloprotease cleavage site.
11. An assay according to claim 1 wherein the substrate comprises:
MAPKK1,
MAPKK2, or
a fragment thereof comprising a metalloprotease cleavage site.
12. An assay according to claim 1 , further comprising washing the solid phase and detecting product on the solid phase.
13. An assay to detect a plurality of metalloproteases in a sample, comprising:
contacting the sample with at least one substrate, wherein each of the plurality of metalloproteases reacts with a substrate of the at least one substrate to form a respective product comprising a tag, and wherein each product is distinguishable from other respective products by mass; followed by
selectively binding the respective tags to a solid phase, wherein the solid phase comprises a binding partner for each respective tag; and
measuring the mass of the respective products, to determine the presence of each of the plurality of the metalloprotease in the sample.
14. An assay according to claim 13 wherein each of the plurality of metalloproteases cleaves the substrate to form a corresponding product having a lower mass than the substrate.
15. An assay according to claim 13 wherein the tag comprises a peptide sequence which binds to a hydrophobic surface.
16. An assay according to claim 13 wherein the tag binds to an antibody.
17. An assay according to claim 13 wherein the tag binds to biotin or streptavidin.
18. An assay according to claim 13 , wherein the substrate is cleaved by botulinum toxin, anthrax toxin or anthrax lethal factor.
19. An assay according to claim 13 , wherein the substrate comprises:
(a) VAMP,
(b) SNAP-25,
(c) syntaxin,
(d) a fragment of any of (a)-(c) comprising a metalloprotease cleavage site, or
(e) a metalloprotease cleavage site.
20. An assay according to claim 13 wherein the substrate comprises:
MAPKK1,
MAPKK2, or
a fragment thereof comprising a metalloprotease cleavage site.