AU4230785A

AU4230785A - Detector kit for the indication of nerve gases and other cholinesterase-inhibitors

Info

Publication number: AU4230785A
Application number: AU42307/85A
Authority: AU
Inventors: Sture Bergek; Erik Dahlgren; Kaj Martensson
Original assignee: Individual
Current assignee: Individual
Priority date: 1984-04-02
Filing date: 1985-04-02
Publication date: 1985-11-01
Also published as: WO1985004424A1; SE8401798D0; SE8401798L; EP0177600A1; JPS61501679A

Description

Detector kit for the indication of nerve gases and other cholinesterase-inhibitors

!

The present invention relates to a detector kit for the indication of nerve gases and other cholinesterase-inhibitors.

It is previously known from U.S. patent No 3,049,411 to indicate nerve gases and other cholinesterase-inhibitors by drawing air through a wet¬ ted filter paper which has been previously treated with choLinesterase. Then a developing reagent, a substrate, is brought in contact with the paper, and a colouring appears if a cholinesterase-inhibitor is not present. Otherwise the colouring reaction does not occur. The substra- te consists of indoxyl acetate or an indophenyl acetate. The colouring reaction occurs as the result of the cholinesterase catalyzing the hydrolysis of the substrate. If a cholinesterase-inhibitor is present, the choLinesterase is inhibited so that it does not catalyze the hydrolysi

Previous indicating systems based on this reaction have all showed dis¬ advantages. There are examples of Large automatic devices with air pumps drawing air past test strips. In Swedish patent No 314,041 there is illustrated a method of exposing a paper having a substrate layer to the influence of air, and then dropping a aqueous solution of choLinesterase onto the substrate. A manually handled detector kit comprising several parts is also known. On a short stick there is a test paper with cholinesterase. First the paper is moistened, and then it is exposed to the influence of air. After that the paper is pressed towards a substrate surface, and the colour change is observed.

As a difference to the cumbersome, automatic test equipments known in the art and the manually handled detector kits consisting of several parts, the present invention relates to a detector kit in one part, which is extremely simple to use and which can be produced at a Low cost and with simple means.

The invention is primarily meant to be used in a war situation where nerve gases might be used. The person, who is going to use the equip¬ ment, may in many cases be under a very strong physical and psychical pressure, the light conditions may be very poor and moreover, this person is wearing a protective mask and a protective suit which among other things involves the use of thick and clumsy gloves. With this as a background, it is very easy to realize that the most possible im¬ portance must be laid upon manageability when there is the question of an equipment meant to be used for the indication of nerve gases.

Thus, the equipment should be of an easily manageable size and be so devised that the indication can be performed with simple movements of one's hand. It is consequently disadvantageous with an equipment which consists of several small parts that are to be moved about during the indication. Moreover, the equipment should be so constructed that one can easily bring the enzyme and the substrate in contact with each other" during the indication.

Besides, it is necessary to store the substrate and the enzyme in an airtight mode. Hitherto known, manually handled indication equipments have all had in common that they have an outer wrapping, which must in some way be broken before the equipment can be 'taken out and used. This operation contributes in lessoning the manageability of the equip- ment, and it must therefore for many applications be looked upon as a great advantage if this outer wrapping could be avoided. Besides, this separate wrapping makes the production more expensive. In more advanced embodiments of the present invention, the detector kit can in a folded form keep the substrate and the enzyme away from the ambient air without using a separate outer wrapping.

As already stated, the enzyme must be wetted for the indication. In prior art manual handling procedures, the enzyme surface has been wetted by means of especially provided water before the indication. However, one cannot be sure to find natural water at the right moment. And it is cumbersome to be forced to have a separate container with water. In some embodiments of the present invention the detector kit is therefore near the enzyme provided with a container comprising water to wet the choLinesterase. The container can be made to burst by means of the pressure from a finger. Besides water the container may comprise a freezing point depressing means, for instance glycol, in order to make the detector kit usable in the cold. It is of very great practical importance that the individual user does not have to start looking for water when he wants to use the detector kit.

It is also important that the liquid container is protected from un¬ intentional puncturing during storage and transport. If the container has been punctured, this must be recognizable when the wrapping is broken. It is here of great importance if one can, not only visually, verify that the enzyme has been wetted, as this may be difficult during troublesome conditions. In some embodiments of the invention the liquid container is so placed on the detector kit that it is broken and becomes loose ;after being punctured and thus clearly shows in this way that it has been punctured.

For water indication there is no need for a wetting agent and it is also possible that so users prefer to do without a wetting agent, for instance for financial reasons. Also because of this, it might be suitable from production point of view to make the liquid container as a separate unit, which is pressed on to the detector kit and comes loose after being used.

The above advantages are obtained with the present invention by giving it the characteristics that are evident from the subsequent patent claims.

In the following the invention will be described in more detail with reference to the attached drawings, where: fig 1a shows a basic embodiment of the invention, fig 1b is a sectional view taken on the Line A-A of fig 1a, fig 2 shows an embodiment of the invention in a Longitudinal sectio¬ nal view with a liquid container close to the enzyme, ig 3a shows another embodiment of the invention in a longitudinal sectional view with a liquid container close to the enzyme, ig 3b shows the invention of fig 3a in a cross-sectional view through the enzyme section, ig 4 shows an embodiment of the invention with a three-section detec¬ tor kit, which can be folded twice and is provided with a liquid container, fig 5a shows an embodiment of the invention which is airtight per se during storage, fig 5b is a sectional view taken on the line B-B of fig 5a with the detector kit folded and provided with a desiccant and a sepa¬ rating foil, fig 6a shows still another embodiment of the invention which is air¬ tight during storage, and fig 6b is a longitudinal sectional view taken on the line C-C of fig 6a.

On a support 1 there are placed the enzyme choLinesterase 2 and also a substrate 3 for the enzyme. The choLinesterase can suitably be immobi¬ lized and stabilized on paper.

i

In a preferred way of producing enzyme paper the following steps are taken. A chromatography paper of the type Whatman DE81 is equilibrated with a 1 M solution of NaCl for 30 minutes, carefully washed with dis¬ tilled water and then allowed to dry at room temperature. On suitably large pieces of this paper, which are cut or punched out from the paper, there is applied 5 μl of an enzyme solution in 0.1 M phosphate buffer CpH = 7.4) consisting of choLinesterase from plaice, saccharose (160 mg/ml), dextran T 10 (160 mg/ml) and as a wetting agent Tween 80 (4 μl/ l). The enzyme papers prepared in this way are dried, first in ambient air and then in an exsiccator under vacuum.

Throughout the tests an enzyme has been used with a specific activity of 0.83 μmol/min/mg. The amount of enzyme in the enzyme solution has been varied between 20 and 80 mg/ml. 40 mg/ml has proved to be a suitable amount when it comes to tackle storage and indication success¬ fully.

The substrate, which has proved to have the best properties for an en¬ zymatic colour reaction, is 2,6-dichloro indophenyl acetate (DCIPA). In the synthesis of this substance one has previously acetylated 2_;6- dichloro indophenol with a large excess of acetic anhydride. In a preferred way of producing DCIPA in connection with the invention, the working up has been modified in as much, as DCIPA has been precipitated in water and then recrystallized in n-hexane:diethyl ether.

As a carrier for DCIPA the following paper qualitites have proved to be suitable: the chromatography paper Whatman No 1 and three filter papers from Munktell with the designations 00H,00M and IF.

The substrate paper is produced by dissolving DCIPA in a suitable soL- vent, e.g. acetone, carbon tetrachloride, chloroform, dichloromethane or 1,2-dichloroethane, and then applying the solution in drops onto the paper. So much of the.solution is used that the whole paper is evenly

2 moistened. The surface concentration, i.e. the amount of DCIPA per cm , is varied by changing the concentration of DCIPA in the solution.

The following surface concentrations have been tested: 80 μg/cm ,

2 2 2

40 μg/cm , 20 μg/cm and 13 μg/cm .

It has been found that the surface concentration is not critical as long as a sufficiently Large amount of the substrate is available in order to get a clear change in the enzymatic colour reaction. Be¬ cause of the losses that one must take into account during storage, in the form of decomposition and possibly sublimation of the substrate,

2 the higher surface concentration of 80 μg/cm is recommended.

As to the support 1, various plastic materials have been tested with regard to the possibility of heat-welding paper thereto and with re¬ gard to how well it can be stored together with DCIPA.

The Latter test has been accomplished by placing paper, impregnated with DCIPA, between two pieces of the plastic in question. The whole package has then been stored in heat at + 60 C together with a de- siccant. After 46 days the sample was taken out and examined with re¬ gard to whether DCIPA had diffused into the plastic or not and with regard to how well the DCIPA-papers functioned during indication.

The following plastic materials are considered usable for the pro- duction of detector kits using enzyme: Plexiglas,. polystyrene and polycarbonate.

In figs 2, 3a, 3b, 5b and 6b is illustrated how a liquid container 4 can be placed close to the enzyme. The container is provided with a pin 5, which perforates the support 1 when a finger is pressed there- on_; so that water will pass to the enzyme. It is also possible with other ways of arranging a Liquid container close to the enzyme.

In figs 3a and 3b is illustrated how the water container 4 can be placed on the support 1 by means of a casing 10 pressed on to the support. The casing is hard and brittle and is cracked on use, where¬ after it loosens from the support 1 thus giving a clear indication of the fact that the enzyme has been wetted. (Important for instance if wetting should occur unintentionally during storage.) Besides, the sharp cracking sound and the detachment from the support give clear signals during correct handling that the enzyme has become satisfac¬ tory wetted.

In fig 4 is illustrated a detector kit in three sections with the en¬ zyme 2 on the middle section. A liquid container 4 is placed on a second section of the detector kit. By folding this section of the detector kit over the enzyme 2, which can be provided with pins 5, the container 4 can be made to break so that water will wet the enzyme. On the third section of the detector kit the substrate 3 is placed. After the wetting of the enzyme the second section is unfolded, and the third section is folded over the enzyme so that the enzyme and the substrate are brought into contact with each other.

In figs 5a and 5b is illustrated an embodiment of the invention with a sealing device 6 around the substrate and the enzyme. When the detec¬ tor kit is being folded during storage, an airtight space is thus ob¬ tained. The sealing device consists of raised edges arranged on the support and extending aroung the enzyme and the substrate and of such dimensions that, when the detector kit is folded, one of the raised edges will come into a firm engagement with the other raised edge. Thus, the two edges can have such a cross-section that they will snap into each other. In many cases, however, a sufficient sealing is obtained already with straight edges. In the simplest form the raised edges are circular but other forms can of course be used. In use, the de- tector kit is pulled apart, before or after the enzyme is wetted, and is exposed to the influence of air, and then the enzyme and the sub¬ strate are brought together.

In figs 6a and 6b is illustrated another embodiment, which also is airtight per se during storage. The edges 7 of the support for the enzyme and the substrate are here welded together or sealingly faste¬ ned to each other in another way, so that a folded detector kit is being formed comprising an airtight space in which the substrate and the enzyme are located. In use, the detector kit is torn apart, and then the procedure is continued as described above.

In the two embodiments described last, the enzyme and the substrate are kept apart during storage by means of. a foil 8 over the substrate. In this way the substrate is prevented from diffusing over to the enzyme. The foil can be torn away after the detector kit has been opened or be so fastened over the' substrate that it is automatically torn away when the detector kit.is opened. A thin aluminium foil has been used with a good result.

As an airtight separate cover for the embodiments of the detector kit, which are not airtight per se, there has all through the tests been

2 used a triple laminate consisting of polyester 18 g/m , Al-foil 0.009 mm and polyethylene 0.07 mm. This foil has been found to have the de¬ sired qualities when it comes to sealing and storage.

In order to make DCIPA stabile during storage it has been found neces¬ sary to have a desiccant 9 in the package. Silica gel and molecular sieve (4 A) have both functioned well. Any difference between the de- siccants havenot been noticed, when it comes to stabilize DCIPA.

Claims

Claims:

1. Detector kit for indication of nerve gases and other cholinesterase- inhibitors consisting of a support (1). on which are provided a surface (2) with choLinesterase and a substrate surface (3) for the enzyme, cha¬ racterized in that the detector kit is foldable so that the surfaces can be brought in abutting engagement with each other.

2. Detector kit according to claim 1, characterized in that there is provided on the detector kit a container (4) comprising water and arran¬ ged to break at an outer pressure and thus wet the enzyme (2).

3. Detector kit according to claim 2, characterized in that the con¬ tainer (4) also comprises a freezing point depressing means. J

4. Detector kit according to claim 2 or 3, characterized in that the water container (4) is placed close to the enzyme (2).

5. Detector kit according to claim 4, characterized in that the water container (4) is located in a brittle casing (10), which is pressed on to the detector kit and cracks at an adapted, outer pressure, whereby the casing falls off the detector kit and thus gives a clear indication that the enzyme (2) has been wetted.

6. Detector kit according to claim 2 or 3, characterized in that the water container (4) is placed on a particular section of the detector kit, which section can be brought into contact with the enzyme (2) by folding the detector kit along a zone, forming a joint and devised for this purpose.

7. Detector kit according to any one of the claims 1-5, characterized in that around the substrate (3) and around the enzyme (2) there is pro¬ vided on the support a raised edge (6), which raised edges will get in¬ to a firm engagement with each other when the detector kit is folded, thus forming an airtight space.

8. Detector kit according to claim 7, characterized in that the raised edges (6) have such cross-sections that they will snap into each other when the detector kit is folded.

9. Detector kit according to any one of claims 1-5, characterized in that the detector kit is kept folded during storage, whereby the edges (7) of the support (1) are sealiπgly but breakabLy fastened to each other by means of welding or the like, in which way there is formed an airtight space for the enzyme (2) and the substrate (3).

10. Detector kit according to any one of claims 7-9, characterized in that a foil (8) separates the enzyme (2) from the substrate (3) during storage.