Support for the determination of enzyme activities and process

• US 4,277,561 A
• Filed: 02/16/1977
• Issued: 07/07/1981
• Est. Priority Date: 02/19/1976
• Status: Expired due to Term

• Current Assignee: Laboratoire De Recherche Api
• Original Assignee: Laboratoire De Recherche Api
• Inventors: Monget, Daniel, Nardon, Paul
• Primary Examiner(s): Wiseman, Thomas G.
• Application Number:
  US05/769,277
• Time in Patent Office: 1,602 Days
• Field of Search: 195/99, 195/103.5, 195/127, 23/230 B, 23/253 TP, 424/2, 424/7, 424/8, 424/9, 424/12, 424/, 435/4, 435/14, 435/18, 435/19, 435/21, 435/23, 435/24, 435/29, 435/805, 435/301, 435/287, 422/56, 422/57, 422/58, 422/99
• US Class Current: 435/14
• CPC Class Codes: C12M 21/18 Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• 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
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick
• CPC Class Codes: Y10S 436/826 Additives, e.g. buffers, diluents, preservatives

Abstract

A support and method for the determination of enzyme activity in a biological extract is provided wherein the support comprises a fibrous material impregnated with a substrate and a water-soluble pH stabilizer comprising a first reagent suitable for a pH of enzyme activity over 7 or a second reagent suitable for a pH of enzyme activity of 7 or less; the viscose fibers are sufficiently loose in texture so as to be accessible both to products in solution and to tissuey and cellular mash or whole cells and sufficiently fine to permit adsorption and uniform distribution of reagents over the area thereof.

The method comprises:

(a) preparing a biological extract of the enzyme to be determined;

(b) depositing a sample thereof on the support;

(c) incubating said sample; and

(d) placing on the support a developing reagent comprising a wetting agent and a buffer, and a coloring agent whereby the reactions are brought to a fixed pH, said pH being optimal for the colorimetric reaction between the substrate and the coloring agent.

Claims

1. A support for the determination of enzyme activity in a biological extract comprising a fibrous material impregnated with a substrate labeled with a naphthol and a buffer solution selected from the group consisting of a first buffer solution suitable for a pH of enzyme activity over 7 and a second buffer solution suitable for a pH of enzyme activity of 7 or less and free from color developing agents;

said fibrous material being loose in texture so as to be accessible both to products in solution and to said biological extract consisting of tissuey and cellular mash or whole cells and sufficiently fine to permit adsorption and uniform distribution of reagents over the area thereof; and

said naphthol substrate originally being colorless or pale yellow so that any subsequent coloration of the substrate, which results from coupling of said naphthol released therefrom with a diazonium salt color-developing agent to indicate the presence of enzyme activity, is readily apparent.

2. The support of claim 1 wherein said fibrous material is a sheet of viscose fibers.

3. The support of claim 1 wherein the first buffer solution suitable for a pH of enzyme activity over 7 is tris-HCl.

4. The support of claim 1 wherein the second buffer solution suitable for a pH of enzyme activity of 7 or less is tris-sodium maleate.

5. An apparatus for determining enzyme activity in a biological extract comprising:

(a) a tray having a plurality of cups contained therein; and

(b) a plurality of discs located in the bottom of said cups, said discs being composed of a fibrous material impregnated with a substrate labeled with a naphthol and a buffer solution selected from the group consisting of a first buffer solution suitable for a pH of enzyme activity over 7 and a second buffer solution suitable for a pH of enzyme activity of 7 or less, and free from coloring agents;

said fibrous material being loose in texture so as to be accessible both to products in solution and to biological samples of tissuey and cellular mash or whole cells and sufficiently fine to permit adsorption and uniform distribution of reagents over the area thereof and said naphthol substrate originally being colorless or pale yellow so that any subsequent coloration of the substrate, which results from coupling of the naphthol released therefrom with a diazonium salt color-developing agent to indicate the presence of enzyme activity, is readily apparent.

6. The apparatus of claim 5 wherein said fibrous material is a sheet of viscose fibers.

7. The apparatus of claim 5 wherein the first buffer solution suitable for a pH of enzyme activity over 7 is tris-HCl.

8. The apparatus of claim 5 wherein the second buffer solution suitable for a pH of enzyme activity of 7 or less is tris-sodium maleate.

9. A method of determining enzyme activity comprising:

(a) preparing a biological extract of the enzyme whose activity is to be determined;

(b) depositing a sample of said extract on a support comprising a fibrous material impregnated with a substrate labeled with a naphthol and a buffer solution selected from the group consisting of a first buffer solution suitable for a pH of enzyme activity over 7 and a second buffer solution suitable for a pH of enzyme activity of 7 or less, and free from color-developing agents;

said fibrous material being loose in texture so as to be accessible both to products in solution and to biological samples of tissuey and cellular mash or whole cells and sufficiently fine to permit adsorption and uniform distribution of reagents over the area thereof;

said substrate originally being colorless or pale yellow so that any subsequent coloration of the substrate, which results from coupling of the naphthol released therefrom with a color-developing agent to indicate the presence of enzyme activity, is readily apparent;

(c) incubating said sample;

(d) depositing on said support a developing reagent comprising a wetting agent, a buffer suitable for use at the desired pH and a diazonium salt color-developing agent for coupling with said released naphthol whereby the reactions are brought to a pH optimal for the colorimetric reaction between said substrate and said color-developing agent;

(e) photochemically decomposing any unreacted color-developing agent; and

(f) comparing said color reactions to a color scale indicative of the activity of the enzyme.

10. The method of claim 9 wherein said fibrous material is a sheet of viscose fibers.

11. The method of claim 9 wherein the first buffer solution for a pH of enzyme activity over 7 is tris-HCl.

12. The method of claim 9 wherein the second buffer solution suitable for a pH of enzyme activity of 7 or less is tris-sodium maleate.

13. The method of claim 9 wherein the developing reagent contains a surface-active agent tending to favor release of naphthol retained by said fibers and to solubilize a granular complex formed by said diazonium salt.

14. The method of claim 9 wherein the diazonium salt coloring agent is Fast Blue BB, said agent present in the reagent in proportions of 35 mg to 10 ml of reagent.

15. The method of claim 9, further comprising treating said support after addition of said reagent so as to destroy excess diazonium salt molecules without affecting the coloration of the azo dye complex formed, thereby permitting photometric determination of enzyme activity by reflectometry.

16. The method of claim 9 wherein the developing reagent consists of an aqueous solution of:

tris-(hydroxymethyl)-aminomethane;

37% hydrochloric acid; and

lauryl sulfate.