Method for determining ACE2 activity
View Patent ↗Methods of determining ACE2 activity in a sample by reacting the sample with ACE2-binding units immobilized on a solid carrier and specific for a part of ACE2 not involved in the catalytic activity of ACE2. The reaction provides a signal that can be correlated with the ACE2 activity. These methods allow for ACE2 activity in complex solutions, such as bodily liquids or culture supernatants, to be quantitatively determined using the endogenous activity of the enzyme to be quantified by converting a signal-providing substrate.
1. A method of determining Angiotensin-converting Enzyme II (ACE2) activity, comprising:
providing ACE2-binding units immobilized on a solid carrier and specific only for a part of ACE2 which is not involved in the catalytic activity of ACE2;
contacting the immobilized ACE2-binding units with a sample which potentially contains ACE2, wherein the ACE2 is bound by the ACE2-binding unit;
removing non-binding portions of the sample from the ACE2 bound to the ACE2-binding units;
adding a substrate of ACE2 which is converted by the ACE2 activity, with the conversion providing a signal; and
measuring a change in the signal during a specific period of time, wherein the change is correlated with the ACE2 activity.
2. The method of claim 1 , wherein the signal is a change in extinction or fluorescence.
3. The method of claim 1 , wherein the substrate has a fluorescent part and a fluorescence-quenching part, which can be separated by the ACE2 activity.
4. The method of claim 1 , wherein the ACE2 is membrane-bound ACE2 (mACE2) or secreted ACE2 (seACE2).
5. The method of claim 1 , wherein the ACE2-binding unit is specific for a partial sequence of at least 4 consecutive amino acids of the 373 N-terminal amino acids of ACE2 or of the 362 C-terminal amino acids of ACE2.
6. The method of claim 5 , wherein the ACE2-binding unit is specific for a partial sequence of at least 6 consecutive amino acids of the 373 N-terminal amino acids of ACE2 or of the 362 C-terminal amino acids of ACE2.
7. The method of claim 6 , wherein the ACE2-binding unit is specific for a partial sequence of at least 8 consecutive amino acids of the 373 N-terminal amino acids of ACE2 or of the 362 C-terminal amino acids of ACE2.
8. The method of claim 1 , wherein the ACE2-binding unit is specific for a part of at least 4 amino acids of the ACE2 which have a minimum distance of 0.5 nm from the active center.
9. The method of claim 8 , wherein the ACE2-binding unit is specific for a part of at least 6 amino acids of the ACE2 which have a minimum distance of 0.5 nm from the active center.
10. The method of claim 9 , wherein the ACE2-binding unit is specific for a part of at least 8 amino acids of the ACE2 which have a minimum distance of 0.5 nm from the active center.
11. The method of claim 8 , wherein the ACE2-binding unit is specific for a part of at least 4 amino acids of the ACE2 which have a minimum distance of 0.6 nm from the active center.
12. The method of claim 11 , wherein the ACE2-binding unit is specific for a part of at least 4 amino acids of the ACE2 which have a minimum distance of 0.8 nm from the active center.
13. The method of claim 12 , wherein the ACE2-binding unit is specific for a part of at least 4 amino acids of the ACE2 which have a minimum distance of 1.0 nm from the active center.
14. The method of claim 1 , wherein the ACE2-binding units are antibodies, antibody fragments, and/or antibody derivatives.
15. The method of claim 14 , wherein the antibodies, the antibody fragments, or the antibody derivatives, are polyclonal.
16. The method of claim 15 , wherein the antibodies, antibody fragments, or antibody derivatives specific for the active centers of the ACE2 are depleted to below 20%.
17. The method of claim 16 , wherein the antibodies, antibody fragments, or antibody derivatives specific for the active centers of the ACE2 are depleted to below 1%.
18. The method of claim 14 , wherein the antibodies, the antibody fragments, or antibody derivatives are monoclonal.
19. The method of claim 1 , wherein the substrate is a low-molecular peptide with a length of from 1 to 30 amino acid and the peptide is cleavable by ACE2, with the fluorescence part covalently bound to an expected cleavage product and the quencher is covalently bound to a different expected cleavage product.
20. The method of claim 19 , wherein the quencher is covalently bound to Mca-Ala-Pro-Lys(Dnp)-OH.
21. The method of claim 19 , wherein the substrate is a low-molecular peptide with a length of from 1 to 20 amino acid and the peptide is cleavable by ACE2.
22. The method of claim 21 , wherein the substrate is a low-molecular peptide with a length of from 1 to 10 amino acid and the peptide is cleavable by ACE2.
23. The method of claim 1 , wherein the sample contains chromophoric and/or fluorescent contaminations.
24. The method of claim 1 , wherein the sample is a bodily fluid, homogenized fluid tissue sample, or cell-culture supernatant.
25. The method of claim 24 , wherein the sample is whole blood, serum or a blood sample.
26. The method of claim 1 , further defined as a method of in vitro determination of ACE2 titers in whole blood, serum, or plasma samples.