 |
|
|
|
|
|
 Title: |
EP0518557A2:
Assay method for hydrolytic enzymes[German][French]
[ Derwent Title ]
|
Country:
Kind: |
EP European Patent Office (EPO)
A2 APPLICATION PUBLISHED WITHOUT SEARCH REPORT i
(See also:
EP0518557A3,
EP0518557B1 )
|
| |
| Inventor: |
Heath, William Francis, Jr.;
Lai, Mei-Huei Tsai;
Manetta, Joseph Vincent;
Sportsman, John Richard;
Yan, Sau-Chi Betty;
|
| Assignee: |
ELI LILLY AND COMPANY
News, Profiles, Stocks and More about this company
|
| Published / Filed: |
1992-12-16
/ 1992-06-03
|
| Application Number: |
EP1992000305109
|
| IPC Code: |
Advanced:
C07K 7/22;
C07K 14/16;
C07K 14/78;
C12Q 1/37;
G01N 33/573;
Core:
C07K 7/00;
C07K 14/005;
C07K 14/435;
more...
IPC-7:
C12Q 1/34;
|
| Priority Number: |
|
| Abstract: |
An assay method for the rapid determination of hydrolytic enzyme activity in large numbers of samples is provided which comprises bonding a resin-binding compound, such as biotin, to one side of the scissile bond of the substrate and a reporter molecule, such as a fluorescence marker, to the opposite side of the scissile bond, incubating the modified substrate and the enzyme in multiple well plates, e.g. 96-well plates, optionally in the presence if a test inhibitor or activator compound transferring the incubation solutions to a second multiple well plate having upper and lower chambers separated by a porous membrane the upper chamber of which contains resin beads capable of binding with the resin-binding compound, filtering and washing the wells of the second plate and reading the emission from the plates. The invention also provides protease substrates for HIV-1 protease, vertebrate stromelysin and derivatives thereof which are useful in the assay method.
|
| INPADOC Legal Status: |
Show legal status actions
Family Legal Status Report
|
| Designated Country: |
AT BE CH DE DK ES FR GB GR IT LI LU NL PT SE
|
| Family: |
Show 9 known family members
|
First Claim:
Show all claims |
1. The method for rapidly measuring the activity of a hydrolytic enzyme in multiple samples wherein the substrate for said enzyme comprises enzyme recognition sites on both sides of the cleavage site which comprises
- a) incubating in multiple reaction chambers a hydrolytic enzyme with a substrate for said hydrolytic enzyme wherein said substrate is bonded on one side of the cleavage site with a resin-binding compound and on the opposite side of the cleavage site with a reporter compound;
- b) transferring the incubation solutions from each well of said multiple well plate to the wells of a second multiple well plate wherein the wells have an upper and lower chamber separated by a porous membrane, wherein each upper chamber of said wells contains a solution or suspension of resin beads capable of irreversible binding to said resin-binding compound and, wherein the size of said resin beads precludes passage of the resin bound substrate or the hydrolyzed resin bound portion thereof through said membrane;
- c) Simultaneously filtering and washing each of said two-chambered wells; and
- d) measuring the emission in each well of said second plate.
|
Description
Expand description |
This invention relates to an assay method for determining hydrolytic enzyme activity and to polypeptides and modified polypeptides useful therein. In particular, it relates to a method for determining hydrolytic enzyme activity which is applicable to the rapid screening of large numbers of potential enzyme inhibitors or stimulators.
+ Preparation of Protease Substrates
+ Preparation 1
+ Nα-Biotin-Pro-Gln-Gly-Ile-Ala-Gly-D-Arg-Lys(Nε-FITC)-OH Substrate for Vertebrate Collogenase
+ Preparation 2
+ Nα-Biotin-Gly-Ser-Gln -Asn-Tyr-Pro-Ile-Val-Gly-Lys(Nε-FITC)-OH (SEQ ID NO:4)
+ Substrate for HIV Protease
+ Preparation 3
+ Nα-Biotin-Arg-Arg-Arg-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-Gly-Lys-(Nε-FITC)-OH (SEQ ID NO:5)
+ Substrate for Vertebrate Stromelysin Assay
+ Example 1
+ HIV-1 Protease Inhibitor Assay Procedure
+ Example 2
+ Collagenase Assay Procedure
+ Example 3
+ Stromelysin Assay Procedure
|
