Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~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
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-
-- 4 --
~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
-- 6 --
12S9553
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.
TABLE 1
Stora~e temperature _ Storage time
-30C ~ 5 years
20-25C > 3 years
40C > 1.5 years
62-68C 4 months
*Trade mark
-- 7
12S95S3
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)
TABLE 2
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
12S9553
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.
TABLE 3
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.
-- 10 --
iS3
The enzyme paper sensitivity is evident from table 4.
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.
-- 11 --
~Z59553
TABLE 5
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
- 12 -
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
- 13 -
TABLE 5
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*).
TABLE 6
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
- 14 -
5S3
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.
TABLE 7
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
0
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.
TABLE 8
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
- 15 -
