CA1259553A - Enzyme paper for the indication of cholinesterase- inhibitors, the preparation and use thereof - Google Patents
Enzyme paper for the indication of cholinesterase- inhibitors, the preparation and use thereofInfo
- Publication number
- CA1259553A CA1259553A CA000491908A CA491908A CA1259553A CA 1259553 A CA1259553 A CA 1259553A CA 000491908 A CA000491908 A CA 000491908A CA 491908 A CA491908 A CA 491908A CA 1259553 A CA1259553 A CA 1259553A
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- enzyme
- paper
- cholinesterase
- substance
- chromatography
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Abstract
ABSTRACT
The invention relates to an enzyme paper, which is suit-able for use in the detection of cholinesterase-inhibitors in a gas or liquid, a method of using the enzyme paper for such detec-tion and, a method of making such a paper,which method involves an advantageous immobilization and stabilization of the enzyme on the paper. The enzyme paper consists of an enzyme carrier being a type of chromatography paper with ion exchange capacity and, having immobilized on the carrier, a cholinesterase enzyme, preferably from plaice, at least one component stabilizing the cholinesterase enzyme, preferably a carbohydrate, e.g. sucrose and dextran, and a surface-active substance, e.g. Tween 80*.
*Trade mark
The invention relates to an enzyme paper, which is suit-able for use in the detection of cholinesterase-inhibitors in a gas or liquid, a method of using the enzyme paper for such detec-tion and, a method of making such a paper,which method involves an advantageous immobilization and stabilization of the enzyme on the paper. The enzyme paper consists of an enzyme carrier being a type of chromatography paper with ion exchange capacity and, having immobilized on the carrier, a cholinesterase enzyme, preferably from plaice, at least one component stabilizing the cholinesterase enzyme, preferably a carbohydrate, e.g. sucrose and dextran, and a surface-active substance, e.g. Tween 80*.
*Trade mark
Description
~5~5S3 3970-~0 Tne present invention relates -to an en~yme paper for use in th~ indieatiorl of cholinesterase-inhibitors in, for instance, air or water. The paper exhibits among other things, enhanced sensitivity, storage stabil:ity and wettability. The invention herein also relates to a metllod of making the enzyme paper. The enzyme paper is suitable for detection of nerve gases or other organophosphorous compounds o-~ cholinesterase-inhibiting nature such as, plant control substances, e.g. weed-killers, and plant protectants.
It is previously known that paper impregnated with an enzyme of the cholinesterase type can be used in equipment for detection of nerve gases. Nerve gases inactivate enzymes of this type. This can be illustrated by a colouring reaction normally caused by the enzyme in contact with a suitable substrate. The presence of nerve gas is indicated by the fact that the colour change of the substrate normally caused by the enzyme, fails to occur because of the cholinesterase-inhibiting functiQn of the nerve gas. In the United States patent No. 3,~49,411 there is described the use of a filter paper preimpregnated with the enzyme
It is previously known that paper impregnated with an enzyme of the cholinesterase type can be used in equipment for detection of nerve gases. Nerve gases inactivate enzymes of this type. This can be illustrated by a colouring reaction normally caused by the enzyme in contact with a suitable substrate. The presence of nerve gas is indicated by the fact that the colour change of the substrate normally caused by the enzyme, fails to occur because of the cholinesterase-inhibiting functiQn of the nerve gas. In the United States patent No. 3,~49,411 there is described the use of a filter paper preimpregnated with the enzyme
2~ cholinesterase, which through hydrolysis, decomposes a substrate such as 2,6-dichloro indophenyl acetate or indoxyl acetate with a resulting colour change to blue. The dried filter paper, which has been pretreated with cholesterase and buffered to a pH of 6.4-8.5, is moistened and exposed to the atmosphere that is to be tested and then brought into contact with the substrate in an aqueous solution buffered to a pH of about 6.4-8.5. When exposed to air comprising nerve gas, the enzyme is phosphorylated, and ~Z59553 thus tll* blu* (~olouring will not occur during the subsequent con-tact with the substrate.
However, it has been found that problems exist with known enzyme papers, which have been preimpregnated with the enzyme and dried for later use. There are problems with the ~-tability of the paper during storage and the wettability of the paper during moistening before exposure. According to the Swedish patent No. 314,041, attempts have been made to use a dry enzyme paper for exposure to air born cholinesterase inhibitors.
Cholinesterase, however, has the character of a glue, and a dried impregnation layer can only slowly be influenced by the sample air.
Another problem with enzyme papers of this kind is that the enzyme is easily dissolved out of the paper by water. This ma~es it impossible to use the enzyme paper for the detection of water born cholinesterase-inhibitors. For the same reason, the coloured substrate paper will often become unevenly coloured when used for detection of air born cholinesterase-inhibitors giving rise to unclear results. This is because the enzyme paper must be moistened before use in order to function satisfactorily.
A critical factor in the detection of low concentrations of cholinesterase-inhibitors is the enzyme sensitivity. For instance, existing enzyme papers do not have a totally satisfying lower detection limit to the nerve gas VX. Above all, this limit is dependent on the choice of enzyme. Cholinesterase from plaice has especially good sensitivity qualities as compared to tradi-tionally used enzymes from sources such as bovine serum, electric ~Z59~53 eel or ray. ffowever, the plaice enzyme does not normally have completely satisfactory stability qualities, and this makes the use thereof difficult on conventional enzyme papers.
A possible technique to overcome some of the above problems is to immobilize the enzyme to the paper, i.e. to bind the enzyme chemically or physically to the carrier material by means of covalent, electrostatic or hydro-phobic interaction.
This technique is previously known for other enzymes but not for cholinesterase from plaice.
It is known to a man skilled in the art thàt immobiliza-tion of an enzyme normally means a substantial loss of enzyme activity and, only occasionally results in a stabilization of the enzyme. For instance, this is evident from table 5. It is not to be expected that the sensitivity qualities of the enzyme will be unchanged. Most often, an impaired sensitivity is to be expected.
This is partly because of steric hindrances occurring in the micro-environment of the enzyme and, partly because of the fact that a large quantity of the enzyme is normally inactivated by the immobilization process resulting in a lower specific activity.
Usually enzyme immobilization also means a substantial extra cost for the production of the end product.
A wetting a~ent is often used in other situations in order to improve the wettability of products. However, wetting agents normally have nn inactivating and/or destabilizing effect on the activity of enzymes. Besides, an unsuitable choice of wetting agent can make the immobilization of the enzyme more difficult.
~259553 The present invention relates to an enzyme paper con-sisting of chromatography paper with ion exchange capacity as a carrier of the enzyme, a cholinesterase enzyme immobilized on the carrier; at least one cholinesterase enzyme stabilizing substance;
and a surface active agent. This gives a product with unique qualities which, unexpectedly, are superior in several aspects compared to wha-t a man skilled in the art expect. Above all, what is unique is that the combination of all the good qualities men-tioned above can be obtained at the same time on one paper. I'his combination of good qualities is not previously known and, the enzyme paper is decidedly superior to the corresponding products known up to now.
The present invention also relates to a method of pro-ducing an enzyme paper wherein a solution of the enzyme cholin-esterase, a surface-active agent and at least one cholinesterase enzyme stabilizing substance is applied on a chromatography paper with ion exchange capacity and then, the paper is dried.
The present invention also relates to a method of detecting a substance in a gas or liquid, which substance inhibits the reaction of cholinesterase, which method comprises contacting the enzyme paper as defined in claim 1 with the gas or liquid, and, then contacting the thus-treated enzyme paper with a sub-strate for the enzyme whose colour changes when said substance does not exist in the gas or liquid.
The combination of positive qualities obtained is summarized as follows:
a) The product can be used without any problems for detec-~259S53 tion of both air born and water born cholinesterase inhibitors.b) Contact with the enzyme paper gives an evenly coloured surface of hydrolyzed substrate on paper saturated with the sub-strate, i.e. a distinct indication that cannot be misinterpreted.
c) The sensitivity qualities are better than the qualities of the previously known corresponding products.
d) The wettability of the paper is considerably better than that of previously known enzyme paper.
e) The stability qualities of the enzyme are considerably improved in comparison to free, watersoluble cholesterase from plaice, making the stability of the product better than most of the corresponding enzyme papers existing on the market.
f) A very small loss of enzyme activity occurs because of the immobilization method used, which is essential with respect to sensitivity as well as cost of production.
g) The technique used in the production of the enzyme paper is very simple meaning that -the production cost can be kept suffi-ciently low.
The characteristics of the invention are evident from the subsequent patent claims.
In production of the enzyme paper, use of a type oE
chromatography paper with ion exchanye ~unction, preEerably with anion exchange function and yood mechanica:l wet strength, in com-bination with an enzyme solution comprising,beside the enzyme, a surface-active agent and a stabilizing component, preferably of carbohydrate type, results in an unchanged or insignificantly reduced enzyme activity in the product. The enzyme is thereby lZS9553 immobilized but a]so, an unexpectedly high coupling yield of immobilized enzyme and an unexpectedly high specific activity of the immobilized enzyme is obtained. This is very unexpected, as immobilization methods, normally lead to considerably impaired enzyme activity qualities (see table 5). The sensitivity qualities of the immobilized enzyme are unchanged. At the same time, the we~tability of the enzyme paper is greatly improved. It is very unexpected that the wettability is improved without being accompanied by a decrease in enzyme activity, enzyme stability, coupling yield or sensitivity, as a wetting agent normally in-activates enzymes to some extent. Because of the choice of the immobilization method and the addition of the stabilizing com-ponent, an unexpectedly good enzyme stability in the finished product is achieved. Most other combinations will not give any improvement and, often result in a destabilization.
In a specially suitable embodiment of the invention, Whatman DE 81* chromatography paper of DEAE-cellulose is used. As a wetting agent, a nonionic agent, e.g. Tween 80* is used. The enzyme is preferably cholinesterase from plaice, and the stabil-izing components preferably used are sucrose and a low moleculardextran, e.g. dextran T 10 (Pharmacia Fine Chemicals). The en~yme solution may be buffered, preferably wi~h a phosphate buer.
The substrate, proved to have the best ~ualities for an enzymatic colour reaction of the described type, is 2,6-dichloro iodophenyl acetate (DCIPA). The substrate is suitably applied on an absorbing paper, e.g. chromatography paper or filter paper.
The invention will now be described in more detail in an * Trade mark illustrative but not delimiting way by means of a number of examples.
PROD~CTION OF ENZYME PAPER
Exam~le-1: Enzyme immobiliæed to DEAE-cellulose paper Chromatography paper of the type Whatman DE 81* is equilibrated with a 1 M solution of NaCl for about 30 minutes, carefully washed with distilled water and dried at room temper-ature. On suitably large pieces which are cut or punched out from the paper, e.g. round discs of the diameter 20 mm, there is applied 5 ~1 of an enzyme solution in 0.1 M phosphate buffer (pH 7.4) consisting of cholinesterase from plaice with a specific activity of 1.46 u/mg or 0.83 ~mol/min/mg (40 mg/ml), sucrose (160 mg/ml), dextran T 10 (160 mg/ml) and, as a wetting agent, Tween 80* (4 ~l/ml). The enzyme papers thus produced are air-dried at room temperature and then dried in an exsiccator under vacuum.
Example-2: Storage stability of enzyme papers During storage the enzyme papers are suitably enclosed in a tight package material. In this test, welded polypropylene bags were used and, acceptable storage times are evident from the following table.
Stora~e temperature _ Storage time -30C ~ 5 years 20-25C > 3 years 40C > 1.5 years 62-68C 4 months *Trade mark For comparison, storage tests have been carri.ed out with some alternative combinations of enzyme papers at a storage -temp-erature of 40C. The enzyme papers were produced according to example 1 (where applicable), and the results are evident from table 2, where E = plaice enzyme (0.2 mg)' 1.46 u/mg; 1.62 u/mg protein S = sucrose (0.8 mg) V = dextran T 10 (0.8 mg) T = Tween 80* (0.02 ~1) Paper quality Components Enzyme yield (%) The enzyme half-life (months), i.e. 50%
remaining activity of initial value DEAE-cellulose E, S, D, T 59 > 18 (Whatman DE 81*) (according to the invention " E, S, D 62 >18 " E, T 20 < 1 Cellulose E, S, D, T 45 ~ 5 (Whatman 113)*
From table 2, it is evident that the storage stability is inferior with the Whatman 113* filter paper than with Whatman DE 81*. The latter is a type of chromatography paper with ion exchange function. An immobilization of the enzyme does not occur with a Whatman 113* paper. The results in table 2 show that im-mobilization of the enzyme gives an improved storage stability.
Moreover, the positive effect of the stabilizing components, sucrose and dextran, is demonstrated.
* Trade mark PI~ODUCTION OF SU~3STRATE PAP:~:R
Example-3 2,6-Dichloro indophenol is acetylated with a larg~ ex-cess of acetic anhydride to 2,6-dichloro indophenyl acetate (DCIPA), which is then precipitated in water and recrystallized in n-hexane: diethyl ether.
Whatman 113* paper is impregnated with a saturated solu-6 e~J z ~
tion of DCIPA in ~r~Y-~ (50 ~1 ~e~/cm2 paper), and then the substrate paper is air-dried at room temperature. Suitably large pieces of the substrate paper are punched out.
As a carrier for DCIPA the following paper types have also proved to be suitable: Whatman No. 1* chromatography paper and Munktell 00~, OOM and IF* filter papers.
be~z/~
Besides ~ ~ffeV DCIPA can be dissolved in other sol-vents such as acetone, carbon tetrachloride, chloroform, dichloro-methane or 1,2-dichloroethane. The solution is added dropwise to the paper so that the whole paper becomes evenly moistened. The surface concentration, i.e. the amount of DCIPA per cm2, can be varied by changing the concentration of DCIPA in the solution. It has been found that the surface concentration is not critical as long as a sufficiently large amount of substrate is available in order to give a clear colour change in the enzymatic re~ction.
Preferably, a relatively high surEace concentration is used, e.g. 80-100 ~g/cm2, in order to compensate for losses in the form of decomposition and possibly, sublimation of the substrate during storage.
In order to get an improved stability of DCIPA during *Trade mark _ g _ '1259~iS3 storage for a longer period of time, the substrate paper produced as above should be stored with a desiccant, e.g. silica gel or a molecular sieve (4 A).
DETECTION
Example-4: Detection of cholinesterase-inhibitor in gas -An enzyme paper, produced as described in example 1, is moistened with about 4 drops of distilled water and then, the paper is exposed for 2 minutes to the gas suspected of containing cholinesterase-inhibitors. The paper is developed by pressing a substrate paper towards it for 2 minutes and, the result is eval-uated. An uncoloured, pink or, very faint grey colour on the substrate paper indicates that cholinesterase-inhibitors are present in the tested gas. ~ blue or faint blue colouring indi-cates that no cholinesterase-inhibitors are present. The enzyme paper sensitivity for the indication of sarin, i.e. isopropyl-methyl phosphono fluoridate, and other organo-phosphorous com-pounds that are called nerve gases, is evident from the following table.
Cholinesterase-inhibitor Paper sensitivity (~g/m3) .. . . _ .
Sarin 0-0 vx o oa.
Example-5: Detection of cholinesterase-inhibitor in liquid Enzyme paper produced according to example 1 is not initially moistened but, is dipped while stirring for 2 minutes into an aqueous solution comprising the actual inhibitor. The development of the paper is carried out as described in example 4.
iS3 The enzyme paper sensitivity is evident from table 4.
Inhibitor Paper sensitivity (mg/l) Sarin 0.6 VX 0.05 COMPAR~TIVE TESTS
Example-6 Other methods of immobilizing the enzyme to a carrier were carried out for comparison. It was found that unexpectedly good results were obtained with the method according to the inven-tion with respect to coupling yield (about 60%) in comparison with other techniques (about 0-7%). This can be seen from the following table.
~Z59553 Enzyme activity Activity of immobilized Immobilizing method (u) before preparation immobilization u %
. . . _ _ _ Adsorption to Amberlite XAD 7* 29.2 0.2 0.7 XAD 2* 29.2 0 0 IR 120-A13+* 29.2 0.1 0O3 Adsorption to Hexylsepharose 6 B* 29.2 0.9 3.1 Hexylsepharose 6 B+*
substrate 29.2 0.6 2.1 Hexylcellulose 29.2 0.5 1.7 Whatman 1 PS* 29.2 0 0 Whatman 1 PS*
+ C12H250S03NA 29.2 0 0 Adsorption witn special technique to Whatman SG 81* 5.84 0.4 6.8 Whatman 4 C~R* 5.84 0.2 3.4 Crosslinking with glutaraldehyde to Whatman SG 81* 58.4 1.6 2.7 Whatman GF/B* 58.4 0.8 1.4 Crosslinking with glutaraldehyde, special technique, to Whatman SG 81* 5.84 0.13 2.2 Whatman 4 CHR* 5.84 0.06 1.0 *Trade mark 1~SS3 TA~LE 5 Enzyme activity ~ctivity of immobilized Immobilizing method (u) before preparation immobilization u Covalent bonding by means of 2-amino-4,6 dichlorotriazine to Whatman 4 CHR* 29.2 0.1 0.3 Whatman 4 CHR*
+ substrate 29.2 0.3 1.0 Covalent bonding by means of CNBr to Whatman 4 CHR* 29.2 0.3 1.0 Whatman 4 CHR*
+ substrate 29.2 0.4 1.4 Covalent immobilization by means of glutaral-dehyde to Whatman GF/C* 29.2 0.3 1.O
Whatman GF/C*
+ substrate 29.2 0.3 1.0 Covalent bonding by means of carbodiimide to Whatman GF/C* 23.0 0.62 2.7 Whatman GF/C*
+ substrate 23.0 0.40 1.7 Whatman CM 82* 23.0 0.47 ~.U
Whatman DM 82*
+ sub~trate 23.0 0.56 2.4 *Trade mark Enzyme activity Activity of immobilized Immobilizing method (u) before preparation immcbilization u Adsorption to Whatman DE 81* +
sucrose and dextran T 10 29.2 ~5 60 Whatman DE 81* +
sucrose and dextran 29.2 16.5 57 Whatman DE 81* +
sucrose, dextran and Tween 80* 29.2 17.2 59 Whatman DE 81* +
sucrose and dextran 29.2 18.1 62 Example-7 Enzyme paper produced according to example 1 without the addition of a wetting agent and the detection ability, i.e. the enzyme paper sensitivity, was evaluated. Table 6 shows the threshold for detection of cholinesterase inhibitors in gas and liquid and, on paper with or without a wetting agent (Tween 80*).
Indication threshold Cholinesterase-inhibitor _gaa_(mg/m3) 1 Sarin; with wetting agent0.04 0.~
VX; with wetting agent 0.04 0.05 Sarin; without wetting agent 0.04 0.6 VX; without wetting agent0.04 0.05 From the table it is evident tha-t the addition of the wetting agent does not impair the enzyme paper sensitivity.
* Trade mark I'ests show that the en~yme paper's stability is not impaired by addition of the wetting agent (see table 7). In table 7, relative activity means enzyme activity as a percentage of the activity present at time 0.
Storage temp. Storage time Rel.act.(~) With Tween 80* 40C 5 months68, 88 Without Tween 80* 40C 5 months 67 With Tween 80* 62-68C 0.5 months60, 71 Without Tween 80* 62-68C 0.5 months 65 With Tween 80* 62-68C 5.5 months 27, 20 Without Tween t~-* 62-68C 5.5 months 29 From table 8 it is evident that the addition of a wetting agent does not decrease the enzyme activity with respect to coupling yield as would otherwise be expected.
Enzyme activity in ~ of added enzyme activity .
With Tween 80* 57, 48, 45 Without Tween 80* 41, 44 These tests show that the addition of the wetting agent, Tween 80*, does not influence the enzyme paper in a negative way with respect to activity or sensitivity and, ak the same time, the enæyme paper exhibits an almost perfect wettability being soaked through within the course of a few seconds.
* Trade mark
However, it has been found that problems exist with known enzyme papers, which have been preimpregnated with the enzyme and dried for later use. There are problems with the ~-tability of the paper during storage and the wettability of the paper during moistening before exposure. According to the Swedish patent No. 314,041, attempts have been made to use a dry enzyme paper for exposure to air born cholinesterase inhibitors.
Cholinesterase, however, has the character of a glue, and a dried impregnation layer can only slowly be influenced by the sample air.
Another problem with enzyme papers of this kind is that the enzyme is easily dissolved out of the paper by water. This ma~es it impossible to use the enzyme paper for the detection of water born cholinesterase-inhibitors. For the same reason, the coloured substrate paper will often become unevenly coloured when used for detection of air born cholinesterase-inhibitors giving rise to unclear results. This is because the enzyme paper must be moistened before use in order to function satisfactorily.
A critical factor in the detection of low concentrations of cholinesterase-inhibitors is the enzyme sensitivity. For instance, existing enzyme papers do not have a totally satisfying lower detection limit to the nerve gas VX. Above all, this limit is dependent on the choice of enzyme. Cholinesterase from plaice has especially good sensitivity qualities as compared to tradi-tionally used enzymes from sources such as bovine serum, electric ~Z59~53 eel or ray. ffowever, the plaice enzyme does not normally have completely satisfactory stability qualities, and this makes the use thereof difficult on conventional enzyme papers.
A possible technique to overcome some of the above problems is to immobilize the enzyme to the paper, i.e. to bind the enzyme chemically or physically to the carrier material by means of covalent, electrostatic or hydro-phobic interaction.
This technique is previously known for other enzymes but not for cholinesterase from plaice.
It is known to a man skilled in the art thàt immobiliza-tion of an enzyme normally means a substantial loss of enzyme activity and, only occasionally results in a stabilization of the enzyme. For instance, this is evident from table 5. It is not to be expected that the sensitivity qualities of the enzyme will be unchanged. Most often, an impaired sensitivity is to be expected.
This is partly because of steric hindrances occurring in the micro-environment of the enzyme and, partly because of the fact that a large quantity of the enzyme is normally inactivated by the immobilization process resulting in a lower specific activity.
Usually enzyme immobilization also means a substantial extra cost for the production of the end product.
A wetting a~ent is often used in other situations in order to improve the wettability of products. However, wetting agents normally have nn inactivating and/or destabilizing effect on the activity of enzymes. Besides, an unsuitable choice of wetting agent can make the immobilization of the enzyme more difficult.
~259553 The present invention relates to an enzyme paper con-sisting of chromatography paper with ion exchange capacity as a carrier of the enzyme, a cholinesterase enzyme immobilized on the carrier; at least one cholinesterase enzyme stabilizing substance;
and a surface active agent. This gives a product with unique qualities which, unexpectedly, are superior in several aspects compared to wha-t a man skilled in the art expect. Above all, what is unique is that the combination of all the good qualities men-tioned above can be obtained at the same time on one paper. I'his combination of good qualities is not previously known and, the enzyme paper is decidedly superior to the corresponding products known up to now.
The present invention also relates to a method of pro-ducing an enzyme paper wherein a solution of the enzyme cholin-esterase, a surface-active agent and at least one cholinesterase enzyme stabilizing substance is applied on a chromatography paper with ion exchange capacity and then, the paper is dried.
The present invention also relates to a method of detecting a substance in a gas or liquid, which substance inhibits the reaction of cholinesterase, which method comprises contacting the enzyme paper as defined in claim 1 with the gas or liquid, and, then contacting the thus-treated enzyme paper with a sub-strate for the enzyme whose colour changes when said substance does not exist in the gas or liquid.
The combination of positive qualities obtained is summarized as follows:
a) The product can be used without any problems for detec-~259S53 tion of both air born and water born cholinesterase inhibitors.b) Contact with the enzyme paper gives an evenly coloured surface of hydrolyzed substrate on paper saturated with the sub-strate, i.e. a distinct indication that cannot be misinterpreted.
c) The sensitivity qualities are better than the qualities of the previously known corresponding products.
d) The wettability of the paper is considerably better than that of previously known enzyme paper.
e) The stability qualities of the enzyme are considerably improved in comparison to free, watersoluble cholesterase from plaice, making the stability of the product better than most of the corresponding enzyme papers existing on the market.
f) A very small loss of enzyme activity occurs because of the immobilization method used, which is essential with respect to sensitivity as well as cost of production.
g) The technique used in the production of the enzyme paper is very simple meaning that -the production cost can be kept suffi-ciently low.
The characteristics of the invention are evident from the subsequent patent claims.
In production of the enzyme paper, use of a type oE
chromatography paper with ion exchanye ~unction, preEerably with anion exchange function and yood mechanica:l wet strength, in com-bination with an enzyme solution comprising,beside the enzyme, a surface-active agent and a stabilizing component, preferably of carbohydrate type, results in an unchanged or insignificantly reduced enzyme activity in the product. The enzyme is thereby lZS9553 immobilized but a]so, an unexpectedly high coupling yield of immobilized enzyme and an unexpectedly high specific activity of the immobilized enzyme is obtained. This is very unexpected, as immobilization methods, normally lead to considerably impaired enzyme activity qualities (see table 5). The sensitivity qualities of the immobilized enzyme are unchanged. At the same time, the we~tability of the enzyme paper is greatly improved. It is very unexpected that the wettability is improved without being accompanied by a decrease in enzyme activity, enzyme stability, coupling yield or sensitivity, as a wetting agent normally in-activates enzymes to some extent. Because of the choice of the immobilization method and the addition of the stabilizing com-ponent, an unexpectedly good enzyme stability in the finished product is achieved. Most other combinations will not give any improvement and, often result in a destabilization.
In a specially suitable embodiment of the invention, Whatman DE 81* chromatography paper of DEAE-cellulose is used. As a wetting agent, a nonionic agent, e.g. Tween 80* is used. The enzyme is preferably cholinesterase from plaice, and the stabil-izing components preferably used are sucrose and a low moleculardextran, e.g. dextran T 10 (Pharmacia Fine Chemicals). The en~yme solution may be buffered, preferably wi~h a phosphate buer.
The substrate, proved to have the best ~ualities for an enzymatic colour reaction of the described type, is 2,6-dichloro iodophenyl acetate (DCIPA). The substrate is suitably applied on an absorbing paper, e.g. chromatography paper or filter paper.
The invention will now be described in more detail in an * Trade mark illustrative but not delimiting way by means of a number of examples.
PROD~CTION OF ENZYME PAPER
Exam~le-1: Enzyme immobiliæed to DEAE-cellulose paper Chromatography paper of the type Whatman DE 81* is equilibrated with a 1 M solution of NaCl for about 30 minutes, carefully washed with distilled water and dried at room temper-ature. On suitably large pieces which are cut or punched out from the paper, e.g. round discs of the diameter 20 mm, there is applied 5 ~1 of an enzyme solution in 0.1 M phosphate buffer (pH 7.4) consisting of cholinesterase from plaice with a specific activity of 1.46 u/mg or 0.83 ~mol/min/mg (40 mg/ml), sucrose (160 mg/ml), dextran T 10 (160 mg/ml) and, as a wetting agent, Tween 80* (4 ~l/ml). The enzyme papers thus produced are air-dried at room temperature and then dried in an exsiccator under vacuum.
Example-2: Storage stability of enzyme papers During storage the enzyme papers are suitably enclosed in a tight package material. In this test, welded polypropylene bags were used and, acceptable storage times are evident from the following table.
Stora~e temperature _ Storage time -30C ~ 5 years 20-25C > 3 years 40C > 1.5 years 62-68C 4 months *Trade mark For comparison, storage tests have been carri.ed out with some alternative combinations of enzyme papers at a storage -temp-erature of 40C. The enzyme papers were produced according to example 1 (where applicable), and the results are evident from table 2, where E = plaice enzyme (0.2 mg)' 1.46 u/mg; 1.62 u/mg protein S = sucrose (0.8 mg) V = dextran T 10 (0.8 mg) T = Tween 80* (0.02 ~1) Paper quality Components Enzyme yield (%) The enzyme half-life (months), i.e. 50%
remaining activity of initial value DEAE-cellulose E, S, D, T 59 > 18 (Whatman DE 81*) (according to the invention " E, S, D 62 >18 " E, T 20 < 1 Cellulose E, S, D, T 45 ~ 5 (Whatman 113)*
From table 2, it is evident that the storage stability is inferior with the Whatman 113* filter paper than with Whatman DE 81*. The latter is a type of chromatography paper with ion exchange function. An immobilization of the enzyme does not occur with a Whatman 113* paper. The results in table 2 show that im-mobilization of the enzyme gives an improved storage stability.
Moreover, the positive effect of the stabilizing components, sucrose and dextran, is demonstrated.
* Trade mark PI~ODUCTION OF SU~3STRATE PAP:~:R
Example-3 2,6-Dichloro indophenol is acetylated with a larg~ ex-cess of acetic anhydride to 2,6-dichloro indophenyl acetate (DCIPA), which is then precipitated in water and recrystallized in n-hexane: diethyl ether.
Whatman 113* paper is impregnated with a saturated solu-6 e~J z ~
tion of DCIPA in ~r~Y-~ (50 ~1 ~e~/cm2 paper), and then the substrate paper is air-dried at room temperature. Suitably large pieces of the substrate paper are punched out.
As a carrier for DCIPA the following paper types have also proved to be suitable: Whatman No. 1* chromatography paper and Munktell 00~, OOM and IF* filter papers.
be~z/~
Besides ~ ~ffeV DCIPA can be dissolved in other sol-vents such as acetone, carbon tetrachloride, chloroform, dichloro-methane or 1,2-dichloroethane. The solution is added dropwise to the paper so that the whole paper becomes evenly moistened. The surface concentration, i.e. the amount of DCIPA per cm2, can be varied by changing the concentration of DCIPA in the solution. It has been found that the surface concentration is not critical as long as a sufficiently large amount of substrate is available in order to give a clear colour change in the enzymatic re~ction.
Preferably, a relatively high surEace concentration is used, e.g. 80-100 ~g/cm2, in order to compensate for losses in the form of decomposition and possibly, sublimation of the substrate during storage.
In order to get an improved stability of DCIPA during *Trade mark _ g _ '1259~iS3 storage for a longer period of time, the substrate paper produced as above should be stored with a desiccant, e.g. silica gel or a molecular sieve (4 A).
DETECTION
Example-4: Detection of cholinesterase-inhibitor in gas -An enzyme paper, produced as described in example 1, is moistened with about 4 drops of distilled water and then, the paper is exposed for 2 minutes to the gas suspected of containing cholinesterase-inhibitors. The paper is developed by pressing a substrate paper towards it for 2 minutes and, the result is eval-uated. An uncoloured, pink or, very faint grey colour on the substrate paper indicates that cholinesterase-inhibitors are present in the tested gas. ~ blue or faint blue colouring indi-cates that no cholinesterase-inhibitors are present. The enzyme paper sensitivity for the indication of sarin, i.e. isopropyl-methyl phosphono fluoridate, and other organo-phosphorous com-pounds that are called nerve gases, is evident from the following table.
Cholinesterase-inhibitor Paper sensitivity (~g/m3) .. . . _ .
Sarin 0-0 vx o oa.
Example-5: Detection of cholinesterase-inhibitor in liquid Enzyme paper produced according to example 1 is not initially moistened but, is dipped while stirring for 2 minutes into an aqueous solution comprising the actual inhibitor. The development of the paper is carried out as described in example 4.
iS3 The enzyme paper sensitivity is evident from table 4.
Inhibitor Paper sensitivity (mg/l) Sarin 0.6 VX 0.05 COMPAR~TIVE TESTS
Example-6 Other methods of immobilizing the enzyme to a carrier were carried out for comparison. It was found that unexpectedly good results were obtained with the method according to the inven-tion with respect to coupling yield (about 60%) in comparison with other techniques (about 0-7%). This can be seen from the following table.
~Z59553 Enzyme activity Activity of immobilized Immobilizing method (u) before preparation immobilization u %
. . . _ _ _ Adsorption to Amberlite XAD 7* 29.2 0.2 0.7 XAD 2* 29.2 0 0 IR 120-A13+* 29.2 0.1 0O3 Adsorption to Hexylsepharose 6 B* 29.2 0.9 3.1 Hexylsepharose 6 B+*
substrate 29.2 0.6 2.1 Hexylcellulose 29.2 0.5 1.7 Whatman 1 PS* 29.2 0 0 Whatman 1 PS*
+ C12H250S03NA 29.2 0 0 Adsorption witn special technique to Whatman SG 81* 5.84 0.4 6.8 Whatman 4 C~R* 5.84 0.2 3.4 Crosslinking with glutaraldehyde to Whatman SG 81* 58.4 1.6 2.7 Whatman GF/B* 58.4 0.8 1.4 Crosslinking with glutaraldehyde, special technique, to Whatman SG 81* 5.84 0.13 2.2 Whatman 4 CHR* 5.84 0.06 1.0 *Trade mark 1~SS3 TA~LE 5 Enzyme activity ~ctivity of immobilized Immobilizing method (u) before preparation immobilization u Covalent bonding by means of 2-amino-4,6 dichlorotriazine to Whatman 4 CHR* 29.2 0.1 0.3 Whatman 4 CHR*
+ substrate 29.2 0.3 1.0 Covalent bonding by means of CNBr to Whatman 4 CHR* 29.2 0.3 1.0 Whatman 4 CHR*
+ substrate 29.2 0.4 1.4 Covalent immobilization by means of glutaral-dehyde to Whatman GF/C* 29.2 0.3 1.O
Whatman GF/C*
+ substrate 29.2 0.3 1.0 Covalent bonding by means of carbodiimide to Whatman GF/C* 23.0 0.62 2.7 Whatman GF/C*
+ substrate 23.0 0.40 1.7 Whatman CM 82* 23.0 0.47 ~.U
Whatman DM 82*
+ sub~trate 23.0 0.56 2.4 *Trade mark Enzyme activity Activity of immobilized Immobilizing method (u) before preparation immcbilization u Adsorption to Whatman DE 81* +
sucrose and dextran T 10 29.2 ~5 60 Whatman DE 81* +
sucrose and dextran 29.2 16.5 57 Whatman DE 81* +
sucrose, dextran and Tween 80* 29.2 17.2 59 Whatman DE 81* +
sucrose and dextran 29.2 18.1 62 Example-7 Enzyme paper produced according to example 1 without the addition of a wetting agent and the detection ability, i.e. the enzyme paper sensitivity, was evaluated. Table 6 shows the threshold for detection of cholinesterase inhibitors in gas and liquid and, on paper with or without a wetting agent (Tween 80*).
Indication threshold Cholinesterase-inhibitor _gaa_(mg/m3) 1 Sarin; with wetting agent0.04 0.~
VX; with wetting agent 0.04 0.05 Sarin; without wetting agent 0.04 0.6 VX; without wetting agent0.04 0.05 From the table it is evident tha-t the addition of the wetting agent does not impair the enzyme paper sensitivity.
* Trade mark I'ests show that the en~yme paper's stability is not impaired by addition of the wetting agent (see table 7). In table 7, relative activity means enzyme activity as a percentage of the activity present at time 0.
Storage temp. Storage time Rel.act.(~) With Tween 80* 40C 5 months68, 88 Without Tween 80* 40C 5 months 67 With Tween 80* 62-68C 0.5 months60, 71 Without Tween 80* 62-68C 0.5 months 65 With Tween 80* 62-68C 5.5 months 27, 20 Without Tween t~-* 62-68C 5.5 months 29 From table 8 it is evident that the addition of a wetting agent does not decrease the enzyme activity with respect to coupling yield as would otherwise be expected.
Enzyme activity in ~ of added enzyme activity .
With Tween 80* 57, 48, 45 Without Tween 80* 41, 44 These tests show that the addition of the wetting agent, Tween 80*, does not influence the enzyme paper in a negative way with respect to activity or sensitivity and, ak the same time, the enæyme paper exhibits an almost perfect wettability being soaked through within the course of a few seconds.
* Trade mark
Claims (17)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Enzyme paper consisting of chromatography paper with ion exchange capacity as a carrier of the enzyme; a cholinesterase enzyme immobilized on the carrier; at least one cholinesterase enzyme stabilizing substance; and a surface active agent.
2. Enzyme paper according to claim 1, wherein the cholin-esterase enzyme is isolated from plaice.
3. Enzyme paper according to claim 1, wherein the enzyme carrier consists of a chromatography paper with anion exchange capacity.
4. Enzyme paper according to claims 1, 2 and 3, wherein the enzyme carrier consists of a paper of DEAE-cellulose.
5. Enzyme paper according to claims 1, 2 and 3, wherein the surface active agent is nonionic.
6. Enzyme paper according to claims 1, 2 and 3, wherein the surface active agent is Tween 80*.
7. Enzyme paper according to claims 1, 2 and 3, wherein the stabilizing substance is a carbohydrate.
*Trade mark
*Trade mark
8. Enzyme paper according to claims 1, 2 and 3, wherein the stabilizing substances is a mixture of sucrose and dextran.
9. Enzyme paper according to claims 1, 2 and 3, wherein the enzyme paper comprises a buffer.
10. Enzyme paper according to claims 1, 2 and 3, wherein the enzyme paper comprises a phosphate buffer.
11. A method of producing an enzyme paper according to claim 1, wherein a solution of the enzyme cholinesterase, a surface-active agent and at least one cholinesterase enzyme stabilizing substance is applied on a chromatography paper with ion exchange capacity and then, the paper is dried.
12. A method according to claim 11, wherein a chromatography paper of DEAE-cellulose is equilibrated with a sodium chloride solution, washed, dried and impregnated with a buffered solution comprising the enzyme cholinesterase from plaice, carbohydrate stabilizing substance and a nonionic surface-active agent, and then the paper is dried.
13. A method of detecting a substance in a gas or liquid, which substance inhibits the reaction of cholinesterase, which method comprises:
contacting the enzyme paper as defined in claim 1 with the gas or liquid, and, then contacting the thus-treated enzyme paper with a substrate for the enzyme whose colour changes when said substance does not exist in the gas or liquid.
contacting the enzyme paper as defined in claim 1 with the gas or liquid, and, then contacting the thus-treated enzyme paper with a substrate for the enzyme whose colour changes when said substance does not exist in the gas or liquid.
14. A method according to claim 13, wherein 2,6-dichloro indophenyl acetate or indoxyl acetate is employed as the substrate and the substance in air or water is detected.
15. A method according to claim 13, wherein the substrate is adsorbed on a carrier of porous paper.
16. A method according to claims 13 and 14 wherein the sub-strate is absorbed on chromatography paper or filter paper.
17. A method according to claims 13, 14 and 15, wherein the presence of a cholinesterase inhibiting nerve gas in air or water is detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000491908A CA1259553A (en) | 1985-09-30 | 1985-09-30 | Enzyme paper for the indication of cholinesterase- inhibitors, the preparation and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000491908A CA1259553A (en) | 1985-09-30 | 1985-09-30 | Enzyme paper for the indication of cholinesterase- inhibitors, the preparation and use thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1259553A true CA1259553A (en) | 1989-09-19 |
Family
ID=4131501
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000491908A Expired CA1259553A (en) | 1985-09-30 | 1985-09-30 | Enzyme paper for the indication of cholinesterase- inhibitors, the preparation and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1259553A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109030166A (en) * | 2018-08-23 | 2018-12-18 | 成都众粒生物科技有限公司 | For detecting gel and its application of Organophosphorus and carbamate pesticides class pesticide concentration |
-
1985
- 1985-09-30 CA CA000491908A patent/CA1259553A/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109030166A (en) * | 2018-08-23 | 2018-12-18 | 成都众粒生物科技有限公司 | For detecting gel and its application of Organophosphorus and carbamate pesticides class pesticide concentration |
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