Stabilization of cholinesterase, detector kit using stabilized cholinesterase, and methods of making and using the same

• US 4,324,858 A
• Filed: 06/16/1980
• Issued: 04/13/1982
• Est. Priority Date: 06/16/1980
• Status: Expired due to Term

• Current Assignee: Stanley M. Burnstein
• Original Assignee: Midwest Research Institute
• Inventors: Goodman, Alan, Goodson, Louis H.
• Primary Examiner(s): Hruskoci, Peter A.
• Application Number:
  US06/160,027
• Time in Patent Office: 666 Days
• Field of Search: 23/230 B, 422/56, 422/57, 435/4, 435/20, 435/176, 435/177, 435/179, 435/180, 435/184, 435/188, 435/196, 435/197, 435/805, 435/810
• US Class Current: 435/20
• CPC Class Codes: C12N 9/18 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/46 involving cholinesterase
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The stability of a cholinesterase (particularly a cholinesterase solution impregnated into a porous material and air-dried) can be improved by: (a) buffering the cholinesterase solution with a zwitterionic buffer, e.g. a buffer having a sulfonic acid group and a protonatable amine group, and, preferably, (b) further drying the impregnated, air-dried porous material under a high vacuum (e.g. 0.01 mm Hg or less) at normal ambient temperatures. The most useful porous materials are sheet-like in nature; that is, they have only two major surfaces. An impregnated, sheet-like material of this invention can be used in a cholinesterase inhibitor detector kit. A typical kit of this type provides a simple means for detecting, inter alia, environmental cholinesterase-inhibiting pollutant, e.g. organophosphorous pesticides and nerve agents.

Claims

1. A method for providing a treated porous material impregnated with a substantially stabilized cholinesterase comprising the steps of:

(a) uniformly distributing the cholinesterase through an aqueous medium buffered to a pH greater than about 6 but less than about 9 with a buffering amount of a zwitterionic buffering agent,

(b) impregnating a porous material with the aqueous medium containing said cholinesterase, and

(c) drying the resulting impregnated porous material to remove substantially all of the physically bound water and thereby provide said treated porous material, having substantially no measurable water vapor pressure in a closed system.

2. A method according to claim 1 wherein the cholinesterase is electric eel cholinesterase.

3. A method of making an anticholinesterase detector kit comprising the steps of:

(a) providing a treated porous material according to claim 2,

(b) providing a second, separate porous material impregnated with an ester substrate for the substantially stabilized cholinesterase contained in said treated porous material,

(c) packaging said treated porous material and said second, separate porous material in a package containing a barrier for preventing contact between said treated porous material and said second, separate porous material until detection of an anticholinesterase is attempted.

4. An anticholinesterase detector kit made according to claim 3.

5. A method for detecting environmental cholinesterase-inhibiting amounts of an anticholinesterase with the detector kit of claim 3 comprising the steps of:

(a) exposing said treated porous material to the environment, and

(b) removing said barrier between said treated porous material and said second, separate porous material and bringing said treated porous material and said second, separate porous material into contact.

6. A method according to claim 3, wherein the zwitterionic buffering agent for the cholinesterase is an 0.5 molar solution of a compound selected from the group consisting of 1,4-bis(3-sulfo-2-hydroxypropyl) piperazine and 1,4-bis(3-sulfopropyl) piperazine.

7. A method for detecting the presence of a cholinesterase-inhibiting amount of an ambient cholinesterase inhibitor comprising the steps of:

(a) providing a treated porous material according to claim 2,

(b) exposing said treated porous material to the ambient conditions, and

(c) contacting said treated porous material with an ester substrate for said substantially stabilized cholinesterase.

14. A method of any of claims 1 or 3 in which the porous substrate is an ion exchange paper.

8. A method for providing a treated porous material impregnated with a substantially stabilized electric eel cholinesterase comprising the steps of:

(a) uniformly distributing the cholinesterase through an aqueous medium buffered to a pH greater than about 6 but less than about 9 with a buffering amount of a zwitterionic buffering agent,

(b) impregnating a porous material with the aqueous medium containing said cholinesterase, and

(c) subjecting the resulting impregnated porous material to a partial vacuum for at least 1 hour in a zone of subatmospheric pressure and generally normal ambient temperature;

the pressure in said zone being less than 1.0 mm of mercury;

thereby obtaining said treated porous material.

9. A method according to claim 8 wherein:

(a) said zwitterionic buffering agent comprises a compound comprising a sulfonic acid group and a protonatable amine group,

(b) said porous material is a sheet-like material having only two major surfaces, and

(c) said pressure in said zone is less than 0.1 mm of mercury, and said porous substrate is subjected to said partial vacuum for about 1-48 hours.

10. A method according to claim 9 wherein:

said zwitterionic buffering agent comprises a substantially water soluble compound of the formula ##STR2## wherein A represents a divalent aliphatic, heterocylcic or carbocyclic aliphatic, or aromatic group, and

R¹ and R² are the same or different and represent carbocyclic or heterocyclic aliphatic, aliphatic, or aromatic groups, or R¹ and R² can together constitute the residue of a carbocyclic or heterocyclic cycloaliphatic or aromatic ring.

11. A method according to claim 10 wherein said zwitterionic buffering agent is selected from the group consisting of the substantially water-soluble 1,4-bis(sulfoalkyl) piperazines, 1,4-bis(sulfohydroxyalkyl) piperazines, omega-monoaminoalkyl sulfonic acids, and combinations thereof.

12. A method according to claim 11, wherein the zwitterionic buffering agent for the cholinesterase is an 0.5 molar solution of a compound selected from the group consisting of 1,4-bis(3-sulfo-2-hydroxypropyl) piperazine and 1,4-bis(3-sulfopropyl) piperazine.

13. A method according to claim 10 wherein said aqueous medium buffered with said zwitterionic buffering agent comprises a 0.01-1.0 molar solution of said zwitterionic buffering agent.

15. A method for providing a treated porous material impregnated with a substantially stabilized electric eel cholinesterase comprising the steps of:

(a) uniformly distributing the cholinesterase through an aqueous medium buffered to a pH greater than about 6 but less than about 9 with a buffering amount of a zwitterionic buffering agent,

(b) impregnating a porous material with the aqueous medium containing said cholinesterase, and

(c) subjecting the resulting impregnated porous material to a partial vacuum in a zone of subatmospheric pressure and generally normal ambient temperature;

the pressure in said zone being less than 1.0 mm of mercury;

thereby removing substantially all physically bound water from the resulting dried electric eel cholinesterase, the water vapor pressure of said dried electric eel cholinesterase being substantially unmeasurable in a closed system.

16. An anti-cholinesterase detector kit, said kit consisting essentially of the following components contained within a moisture resistant package:

(a) an extremely dry, impregnated porous material, said porous material being impregnated with cholinesterase and a buffering amount of a zwitterionic buffering agent, said impregnated porous material being dried by subjecting the resulting impregnated porous material to a partial vacuum for at least 1 hour in a zone of subatmospheric pressure and generally normal ambient temperature;

the pressure in said zone being less than 1.0 mm of mercury;

(b) a dry ester substrate for the cholinesterase, said substrate being separated from the cholinesterase by a barrier for preventing cholinesterase inactivation by sublimation of the substrate during storage.

17. A kit of claim 16 in which the porous material is an ion exchange paper.

18. A kit of claim 17 in which the dry impregnated ion exchange paper has been dried so that it has substantially no measurable water vapor pressure in a closed system.

19. A kit of claim 18 in which the cholinesterase and the ester substrate are supported on a common base which can be folded against itself so that the cholinesterase and substrate can contact each other when the barrier that separates them is removed.