Device for determining the activity of enzymes in liquids

• US 5,935,846 A
• Filed: 02/18/1997
• Issued: 08/10/1999
• Est. Priority Date: 06/20/1995
• Status: Expired due to Term

• Current Assignee: Papst Licensing GmbH & Company Kg
• Original Assignee: Bernd Werle, Johannes Schumacher
• Inventors: Werle, Bernd, Schumacher, Johannes
• Primary Examiner(s): Beisner, William H.
• Application Number:
  US08/793,833
• Time in Patent Office: 903 Days
• Field of Search: 435/286.5, 435/287.3, 435/287.9, 435/288.6, 435/288.7, 435/808, 422/70, 422/69, 422/82.05-82.08, 210/198.2
• US Class Current: 435/288.6
• CPC Class Codes: C12M 41/46 of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions

Abstract

A process and device are disclosed to determine the activity of enzymes in liquids in a largely automatic manner. The device for carrying out this process has a column with an chromatographic carrier for treating a measurement sample. The carrier is mixed with a substance capable of binding to an enzyme inhibitor present in the measurement sample and that corresponds to at least one enzyme. A measurement sample supply is associated to one end of the column. A valve/pump arrangement for filling at least one test tube with a carrier and at least part of the measurement sample is connected downstream of the column, in the flow direction of the measurement sample. The carrier is dissociated into cleavage products by the action of the enzyme. The rise in concentration per unit of time of at least one of the cleavage products of the carrier is sensed during an incubation time. As an alternative or supplementary step, the enzyme that corresponds to at least one enzyme inhibitor is extracted by chromatography from a measurement sample to detect enzyme inhibitors in liquids and the thus treated measurement sample is tested for inhibitor concentration and/or activity.

Claims

1. A device for measuring the activity of an enzyme in liquids comprising:

a vessel, a column, said column having a chromatographic carrier having a substance capable of binding an enzyme inhibitor corresponding to said enzyme in a sample, a valve/pump assembly between said column and said vessel for filling said vessel with a substrate and the sample, said sample and said substrate reacting to form a cleavage product, a detector for measuring the increase of the concentration per unit of time of said cleavage product, and a means for enabling the passage of said sample through said column or outside said column into said vessel.

2. A device according to claim 1 wherein the column can be used repeatedly, as there is an excess of the substance corresponding to the capacity of the column.

3. A device according to claim 1 wherein the column is exchangeable.

4. A device according to claim 1 capable of functioning with a column buffer discharged from the column and including a control device for checking the purity of the column buffer, said control device working photometrically.

5. A device according to claim 1 capable of functioning with a column buffer discharged from the column and including an arrangement connected in series to the column for measuring the degree of dilution of the discharged sample caused by the column buffer, said arrangement capable of also measuring the volume of liquids.

6. A device according to claim 1 capable of functioning with a measuring buffer and a column-buffer, wherein the valve/pump assembly admixes the measuring buffer to the said sample and optionally the column buffer and to the substrate in the is as to produce definite experimental conditions.

7. A device according to claim 1 wherein the detector includes a device for measuring fluorescence.

8. A device according to claim 1 further including a means to thermostat the vessel.

9. A device according to claim 1 further including at least one valve associated with the column to pass a buffer as a wash liquid at least through the column and the valve/pump assembly.

10. A device according to claim 1 wherein further including a computer capable of running and controlling the mixing and charging of the vessel and the detection and evaluation of the concentration increase per unit of time of said cleavage product of the substrate.

11. A device for measuring the activity of an enzyme in liquid comprising:

a vessel, a column for treating a sample, said column carrying a chromatographic carrier treated with a substance capable of binding an enzyme inhibitor corresponding to said enzyme in the sample, said column being connected for discharge into the vessel, a substrate source being connected for discharge into said vessel and reaction with said sample thereby releasing a cleavage product, a detector for measuring the increase of the concentration per unit of time in the treated vessel of said cleavage product and an arrangement connected to the column for measuring the degree of dilution of the discharged sample caused by a column buffer.

12. A device according to claim 11 wherein the column can be used repeatedly, as there is an excess of the substance corresponding to the capacity of the column.

13. A device according to claim 11 wherein the column is exchangeable.

14. A device according to claim 11 further including a control device for checking the purity of a column buffer discharged from the column, said control device working photometrically.

15. A device according to claim 11 further including said arrangement capable of also measuring the volume of liquids.

16. A device according to claim 11 capable of functioning with a buffer, wherein the valve/pump assembly admixes a measuring buffer to the said sample and optionally a column buffer in the vessel so as to produce definite experimental conditions.

17. A device according to claim 11 wherein the detector includes a device for measuring fluorescence.

18. A device according to claim 11 further including a means to thermostat the vessel.

19. A device according to claim 11 further including at least one valve associated with to said column to pass a buffer as a wash liquid at least through the column and the valve/pump assembly.

20. A device according to claim 11 further including a computer to run and control the mixing and charging of the vessel and the detection and evaluation of the concentration increase per unit of time of at least one of the cleavage products of the substrate.

21. A device for measuring the activity of an enzyme in liquid comprising:

a vessel, a column for treating a sample, said column being exchangeable and filled with a chromatographic carrier treated with a substance capable of binding an enzyme inhibitor which corresponds to said enzyme in the sample, a valve/pump assembly having a supply tube connected in series to a sample reservoir so as to fill the vessel with a buffer and the treated sample, a substrate source connected to said vessel for discharge of a substrate into said vessel for reaction with said treated sample, thereby releasing a cleavage product, and a detector for measuring the increase of the concentration per unit of time of said cleavage product, further including a control device connected to the column for monitoring the purity of a buffer discharging the sample from the column.

22. A device according to claim 21 wherein said control device works photometrically.

23. A device according to claim 21 wherein the said device is also able to measure the electrolytic conductance of liquids.

24. A device according to claim 21 further including a device for mixing connected in series to the column as to produce a homogenous mixture of the said sample with the column-buffer.

25. A device according to claim 21 wherein the column can be used repeatedly, as there is an excess of the substance corresponding to the capacity of the column.

26. A device according to claim 21 wherein the column is exchangeable.

27. A device according to claim 21 wherein said control device works photometrically.

28. A device according to claim 21 capable of functioning with a column buffer and an arrangement connected in series to the column for measuring the degree of dilution of the discharged sample caused by the column buffer, said arrangement capable of also measuring the volume of liquids.

29. A device according to claim 21 wherein the valve/pump assembly admixes a measuring buffer to the said sample an optionally the column buffer in the vessel so as to produce definite experimental conditions.

30. A device according to claim 21 wherein the detector includes a device for measuring fluorescence.

31. A device according to claim 21 further including a means to thermostat the vessel.

32. A device according to claim 21 further including a valve corrected to said column to pass a buffer as a wash liquid at least through the column and the valve/pump assembly.

33. A device according to claim 21 further including a computer to run and control the mixing and charging of the vessel and the detection and evaluation of the concentration increase per unit of time of at least one of the cleavage products of the substrate.