Patent EA-005092-B1 (EA)
Title: Scintillation proximity assays for aminoglycoside binding molecules
Inventor: CAMPS MONTSERRAT
IPC: C12N15/09
Filed: 2002-06-06
Abstract: 1. A method for identifying and/or quantifying radiolabeled Aminoglycoside Binding Molecules (ABMs) into a sample, said method comprising the following steps: a) preparing a sample comprising at least an ABM and an enzyme; b) allowing said enzyme either to add a radiolabeled group present in the sample to the ABM, or to cleave a radiolabeled group already present in the ABM; c) incubating the sample with one or more solid supports impregnated with a scintillant compound and covered with an aminoglycoside; and d) measuring the emission generated by the scintillating support (s). 2. The method of claim 1 wherein the sample and the support (s) are separated between steps (c) and (d). 3. The method of claim 1 or 2 wherein different samples prepared according to step (a) comprise equal amount of the ABM, equal amount of the enzyme capable either of adding a radiolabeled group present in the sample to the ABM, or of cleaving a radiolabeled group already present in the ABM, and one or more additional compounds. 4. The method of claim 1 or 2 wherein different samples prepared according to step (a) comprise equal amount of the ABM, equal amount of the enzyme capable either of adding a radiolabeled group present in the sample to the ABM, or of cleaving a radiolabeled group already present in the ABM, with or without one or more additional compounds added before or during step (c). 5. The method of claim 1 or 2 wherein different samples prepared according to step (a) comprise equal amount of the ABM, and different mixtures of molecules potentially containing an enzyme capable either to add a radiolabeled group present in the sample to the ABM, or to cleave a radiolabeled group already present in the ABM. 6. The method of claim 1 or 2 wherein different samples prepared according to step (a) comprise equal amount of an enzyme capable either to add a radiolabeled group present in the sample to the ABM, or to cleave a radiolabeled group already present in the ABM, and different mixtures of molecules potentially containing an ABM. 7. An assay for identifying and/or quantifying compounds modulating the interaction between an ABM and an enzyme capable either of adding a radiolabeled group present in the sample to the ABM, or of cleaving a radiolabeled group already present in the ABM, wherein the scintillation proximity signal generated by the supports obtained according to the method of claim 3 is compared. 8. The assay of claim 7 wherein the compounds are potential inhibitors of the enzyme. 9. An assay for identifying and/or quantifying compounds modulating the interaction between an ABM and an aminoglycoside, wherein the scintillation proximity signal generated by the supports obtained according to the method of claim 4 is compared. 10. The assay of claim 9 wherein the compounds aminoglycoside-modifying bacterial enzyme. 11. An assay for identifying and/or quantifying an enzyme capable either to add a radiolabeled group present in the sample to the ABM, or to cleave a radiolabeled group already present in the ABM, wherein the scintillation proximity signal generated by the supports obtained according to the method of claim 5 is compared. 12. An assay for identifying and/or quantifying an ABM, wherein the scintillation proximity signal generated by the supports obtained according to the method of claim 6 is compared. 13. The assay of claims 7 to 12 wherein a compound competing with, or altering, one of the components included in the assay is added. 14. The assay of claims 7 to 13 wherein the scintillating supports are beads. 15. The assay of claims 7 to 14 wherein the aminoglycoside is neomycin. 16. The assay of claims 7 to 15 wherein the ABM is a mono-or polyphosphatated phosphoinositide. 17. The assay of claims 7 to 15 wherein the ABM is an RNA molecule. 18. The assay of claim 16 wherein the enzyme is a phosphoinositide kinase. 19. The use of a scintillating solid support coated with anaminoglycoside for identifying and/or quantifying a radiolabeled aminoglycoside binding molecule into a sample. 20. A kit for identifying and/or quantifyingaminoglycoside binding molecules, enzymes modifying said molecules, or compounds modulating the interaction between ABMs and either enzymes oraminoglycosides a radiolabeled aminoglycoside binding molecule into a sample, comprising a scintillating solid support covered with an aminoglycoside.