Compositions for the detection of protease in biological samples and methods of use therefo
AssigneeOncoimmunin, Inc.
InventorsAkira Komoriya, Beverly S. Packard
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Abstract
The present invention provides for novel reagents whose fluorescence increases in the presence of particular proteases. The reagents comprise a characteristically folded peptide backbone each end of which is conjugated to a fluorophore. When the folded peptide is cleaved, as by digestion with a protease, the fluorophores provide a high intensity fluorescent signal at a visible wavelength. Because of their high fluorescence signal in the visible wavelengths, these protease indicators are particularly well suited for detection of protease activity in biological samples, in particular in frozen tissue sections. Thus this invention also provides for methods of detecting protease activity in situ in frozen sections.
Claims45
A fluorogenic composition for the detection of the activity of a protease, said composition having the formula:
- ##STR21## wherein, P is a peptide comprising a protease binding site for said protease, said binding site consisting of about 2 to about 8 amino acids;
- F.sup.1 and F.sup.2 are fluorophores;
- S.sup.1 and S.sup.2 are peptide spacers ranging in length from 1 to about 50 amino acids;
- n and k are independently 0 or 1;
- and
- C.sup.1 and C.sup.2 are conformation determining regions comprising peptides ranging in length from 1 to about 3 amino acids, said conformation determining regions positioning said fluorophores adjacent to each other with a separation of less than about 100 .ANG.;
- and
- when n is 1, S.sup.1 is joined to C.sup.1 by a peptide bond through a terminal alpha amino group of C.sup.1 ;
- and
- when k is 1, S.sup.2 is joined to C.sup.2 by a peptide bond through a terminal alpha carboxyl group of C.sup.2.
A method of detecting the activity of a protease in a biological sample, said method comprising the steps of:
- contacting said biological sample with a fluorogenic composition having the formula:
- ##STR24## wherein, P is a peptide comprising a protease binding site for said protease, said binding site consisting of about 2 to about 8 amino acids;
- F.sup.1 and F.sup.2 are fluorophores;
- S.sup.1 and S.sup.2 are peptide spacers ranging in length from 1 to about 50 amino acids;
- n, and k are independently 0 or 1;
- and
- C.sup.1 and C.sup.2 are conformation determining regions comprising peptides ranging in length from about 1 to about 3 amino acids, said conformation determining regions positioning said fluorophores adjacent to each other with a separation of less than about 100 .ANG.;
- and
- when n is 1, S.sup.1 is joined to C.sup.1 by a peptide bond through a terminal alpha amino group of C.sup.1 ;
- and
- when k is 1, S.sup.2 is joined to C.sup.2 by a peptide bond through a terminal alpha carboxyl group of C.sup.2 ;
- and
- detecting a change in fluorescence of said fluorogenic composition wherein an increase in fluorescence indicates protease activity.
Dependencies are inferred from the claim text.