Method of quantitative enzyme determination

• US 5,612,178 A
• Filed: 08/24/1992
• Issued: 03/18/1997
• Est. Priority Date: 11/05/1987
• Status: Expired due to Fees

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Thompson, Richard E., Kapsalis, Andreas A., Gordon, Julian
• Primary Examiner(s): Naff, David M.
• Application Number:
  US07/933,971
• Time in Patent Office: 1,667 Days
• Field of Search: 435/4, 435/15, 435/18, 435/25, 435/805, 436/517, 422/56, 422/57, 422/71
• US Class Current: 435/4
• 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: G01N 33/523 the element being adapted for a specific analyte
• CPC Class Codes: Y10S 435/805 Test papers

Abstract

Methods and kits are provided for the quantitative determination of the presence of an enzyme analyte in a sample by determination of the rate of analyte catalyzed reaction on a chromatographic medium. The methods may be used to determine both steady state and non-steady state enzyme reaction kinetics and provide a physical record reflecting the same. In a preferred embodiment, an analyte enzyme in a sample to be analyzed is immobilized at a reaction site on a chromatographic medium, the chromatographic medium is contacted with a solution containing a substrate, the solution is transported on the chromatographic medium to the reaction site where the analyte enzyme catalyzes the reaction of the substrate to produce a detectable reaction product at a rate related to the amount of enzyme present, and the solution and reaction product are transported from the reaction site to a detection region formed by a length of the chromatographic medium downstream from the reaction site. Transport continues until the solution reaches the end of the chromatographic medium or until the quantity of solution is exhausted.

Claims

1. A method for the quantitative determination of the presence of an analyte enzyme in a sample comprising the steps of:

(a) immobilizing the analyte enzyme present in a quantity of sample to be analyzed at a reaction site on a chromatographic medium,

(b) contacting said chromatographic medium with a solution comprising a substrate,

(c) transporting said solution to said reaction site wherein said analyte enzyme catalyzes the reaction of said substrate to produce a detectable reaction product at a rate related to the amount of enzyme present;

(d) transporting said solution and said reaction product from said reaction site to a detection region comprising a length of said chromatographic medium downstream from said reaction site wherein transport continues until said solution reaches the end of said chromatographic medium or until the quantity of solution is exhausted, and wherein the production of said reaction product results in a continuous record of the rate of reaction, and

(e) detecting a signal produced by (i) said substrate, (ii) said reaction product, or (iii) a reactant or product of one or more additional reactions of said reaction product, at a selected site in said detection region.

2. The method according to claim 1 wherein said selected site in the detection region contains said substrate which is consumed or said reaction product which is produced only during steady state reaction of the analyte enzyme.

3. The method according to claim 1 wherein said additional reaction or reactions are catalyzed by means of a catalyst immobilized at a second reaction site.

4. The method according to claim 3 wherein said catalyst is an enzyme.

5. The method according to claim 1 wherein said signal is detected visually or spectrophotometrically.

6. The method according to claim 5 wherein said signal is a color signal which is detected visually.