DE8905074U1 - Test element for agglutination tests - Google Patents

Description

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F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft, Basel/ Switzerland

RAN4090/187

Test electrode for agglutination tests

The invention relates to a test element for agglutination tests according to the preamble of the patent claim

The detection of certain substances, such as drugs, addictive substances, hormones, viral, bacterial and parasitic antigens, as well as substances which may be indicative of the presence of certain diseases (e.g. tumor markers) and the like in body fluids such as urine or blood can be carried out using agglutination reactions which take place in a mixture of an antibody reagent, a reagent with latex coated with a substance or a metabolite of the substance and a body fluid. Since the antibody contained in the antibody reagent only in limited quantities can combine with both the substance or metabolite of the latex and the substance in the body fluid, agglutinations are only formed if, due to the absence of the substance or metabolite in the body fluid, there is a sufficient quantity of antibody to react with the latex particles, so that

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several latex particles can agglomerate to form visually definable agglutinations. In the presence of the substance or metabolite being sought in the body fluid, however, significantly fewer antibodies are available for the agglomeration, since the antibodies form an immune complex with the substance or metabolite being sought in the body fluid, so that no or only a very weak agglutination reaction can take place.

Another type of detection can be carried out using agglutination reactionsa which are carried out in a mixture of a

TO reagent with latex coated with antibodies against the substance to be detected and the body fluid. If there is a sufficient amount of the substance being sought in the body fluid, this promotes a visually definable agglutination, whereas if it is absent, it does not occur. In a similar way, antibodies can also be detected in blood or other body fluids, whereby the corresponding substances or antigens, or antibodies directed against the antibodies being sought, are used as binding partners in the agglutination system.

One procedure for such immunological tests is that the reagent mixture is placed in a test tube and mixed and kept in reaction by shaking and swirling the tube for a period of time. This procedure can lead to different agglutination reactions, depending on the length of time and the intensity with which the test tube is shaken and swirled. In view of the uncertainty associated with this in evaluating the reaction results, it has already been suggested (cf. DE-A-35 37 734 or US-fc-45 96 695) to allow the agglutination reaction to take place in a reaction capillary. This means that manual spinning and shaking of the sample-reagent mixture can be dispensed with, since the agglutination produced by the

The movement of the mixture along the reaction capillary caused by the capillary action ensures that a suitable mixing of the sample with the reagents for the formation of agglutination and a continued movement of the sample-reagent mixture takes place over a certain period of time.

The reaction capillary of this known test device is provided between two transparent glass plates which are kept at a certain constant distance from each other to form a capillary chamber. The capillary effect for the respective sample-reagent mixture is therefore essentially constant along the length of the capillary chamber. This has the disadvantage that under these circumstances the reaction sensitivity and the reproducibility of the reaction results are subject to strong fluctuations. This is the result of the sample-reagent mixture needing different amounts of time to flow through the capillary chamber when the same test is carried out in different ways. An important criterion for achieving reproducible results is therefore maintaining consistent reaction times. Another prerequisite for optimal sensitivity of the agglutination reaction is that a certain period of time, e.g. 3 minutes, is available for the reaction with only minor deviations upwards and downwards.

The invention is therefore based on the object of creating an easily manageable "test element" of the type mentioned at the beginning, which, with an uncomplicated structure and an economical production option, enables the course of a meaningful agglutination reaction in the respective sample-reagent mixture within a reproducible period of time without the influence of external forces, such as manual shaking or spinning movements or thermal convection.

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This object is achieved according to the invention by the features in the characterizing part of patent claim 1. According to this, the test element is characterized by a reaction capillary in which the flow rate of the sample-reagent mixture in an upstream capillary region is greater than along a downstream region.

In a test element according to the invention, a pumping effect is therefore exerted on the sample-reagent mixture in one area (upstream area) of the reaction capillary, which ensures a constant supply and movement of the mixture into and through the other downstream area, which can therefore be designed for optimal conditions for the formation of agglutinations. As described below, the sample-reagent mixture flows from the upstream to the downstream area. Advantageously, the time required for a sample-reagent mixture to flow from the upstream end to the downstream end of the reaction capillary is subject to only very slight fluctuations between different tests for a given dimension of the test element for a specific test liquid. This fulfills an important condition for the highest possible sensitivity of the agglutination reaction. The reliability of the evaluation of the results is thereby significantly increased. Since the reaction capillary has a downstream area in which the flow rate of the sample-reagent mixture is lower than the upstream area, larger clumps of latex particles can occur without the flow of the sample-reagent mixture through the reaction capillary being significantly impaired or even prevented. On the other hand, larger clumps of latex particles make visual test evaluation and handling of the test element easier in practice.

The transition between the upstream and downstream areas of the reaction capillary can be step-like, but a continuous transition is preferred.

In particular, an embodiment of the test element according to the invention is preferred in which the cross-section of the reaction capillary increases continuously in the flow direction of the sample-reagent mixture (e.g. a linear increase in this cross-section over certain segments of the reaction capillary) or at least does not decrease.

According to a preferred development of the invention, the test element can comprise at least one pair of plate-shaped bodies, between which a reaction capillary with a substantially rectangular cross-sectional configuration is formed, the dimension of the cross-section of the reaction capillary in the thickness direction (i.e. the distance between the plate-shaped bodies) being smaller along the upstream region than along the downstream region. Consequently, greater capillary forces prevail in the upstream region than in the downstream region, which results in correspondingly different flow velocities through the reaction capillary. Tests have shown that compliance with a certain flow time of the sample-reagent mixture through the reaction capillary within narrow limits is further promoted if according to another preferred development of the invention, the reaction capillary has a substantially S-shaped course, whereby an intermediate region with a capillary effect is provided between the upstream and downstream regions, which creates a flow rate for the sample-reagent mixture that is equal to or less than that along the upstream and/or equal to or greater than that along the downstream region of the reaction capillary. At the downstream end of the reaction capillary an area can be provided in which the sample-reagent mixture collects after passing through the capillary. This facilitates the observation and evaluation of the agglutination reaction.

The test element can be made up of injection-molded parts which can be produced in a particularly economical manner and which are made from a suitable transparent plastic material. The test element contains upper and lower plates manufactured by injection molding which provide the reaction capillary between them and are connected to one another by welding or gluing. A preferred method provides that the connection of the upper and lower plates to create a reaction capillary of the configuration according to the invention is carried out by ultrasonic welding by forming a welding web on one of the plate-shaped bodies that extends at least longitudinally along the reaction capillary to be formed and a complementary welding groove on the other plate-shaped body with a cross-sectional configuration such that the welding web can only be partially received therein, bringing the plate-shaped bodies together with the welding web and the welding groove engaging, and applying ultrasonic energy to the plate-shaped bodies so that the areas of the welding web in contact with the welding groove melt and this penetrates into the welding groove to a predetermined depth while connecting the plate-shaped bodies in order to create a reaction capillary with a dimension in the thickness direction that is smaller at the upstream end than at the downstream end. In this way, the dimensions of the reaction capillary in the thickness direction can be precisely maintained to achieve the desired different capillary effect. Ultrasonic welding is particularly cost-effective.

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For other developments of the invention, reference is made to the claims.

In a so-called competitive detection method, a certain amount of body fluid, an antibody reagent and a latex coated with reagent are fed one after the other to a receiving and mixing area of the test element according to the invention, where they are mixed together and fed to the reaction capillary. After the sample-reagent mixture has flowed through the reaction capillary, the presence or absence of the substance being sought is determined by visually determining the presence or absence of agglutination reaction products in the collection area of the test element. The antibody can either be part of the reagent or be present in the body fluid, depending on whether a competitive or non-competitive detection method is carried out.

The invention is explained in more detail below using an embodiment and the drawing. They show:

Fig. 1 shows a top view of a test element for agglutination tests according to a preferred embodiment of the invention,
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Fig. 2-4 sectional views along the section lines H-II, IH-III and IV-IV in Fig. 1,

Fig. 5 is a sectional view along the section line v-V in Fig. 1, and

Fig. 6A, 6B fragmentary cross-sectional views on an enlarged scale of the test element showing the position of the plate-shaped bodies before and after their welding.

The test element according to the preferred embodiment of the invention bears the general reference numeral 1 and comprises, as shown, a pair of lower and upper plate-shaped bodies 2, 3 arranged one on top of the other, which are arranged longitudinally

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are firmly connected to one another by web areas 13, which are explained in more detail, e.g. by gluing or welding. On one of the plate-shaped bodies 2 or 3, in particular the upper body, areas are defined which serve to receive, move and collect a sample-reagent mixture for agglutination reactions, which will also be discussed in more detail below.

In particular, an elongated recess is provided on an end section of the test element 1 on the left in Fig. 1, which penetrates the upper plate-shaped body 3 and is covered in a fluid-tight manner by the lower plate-shaped body 2 at the bottom to form a receiving and misting area for the sample and the agglutination reagents. At the other right-hand end section (i.e. in the Sairanel area) of the test elatnent 1, a substantially rectangular, square or other-shaped recess is defined in the lower surface of the upper plate-shaped body 3, which in conjunction with the lower plate-shaped body 2 covering it defines a chamber or area 8 in which the sample-reagent mixture can collect for further observation after flowing through a substantially S-shaped reaction capillary 4 extending between the areas 7 and 8.

The reaction capillary 4 is formed by a groove-shaped recess in the lower surface of the upper plate-shaped body 3, which is covered in a fluid-tight manner by the lower plate-shaped body 2. The web 13 extends along the receiving and mixing area 7, the reaction capillary 4 and the screening area 8, thus forming a lateral fluid-tight boundary of these areas and the reaction capillary (see Fig. 2-4).

The reaction capillary 4 comprises a first or upstream capillary region 9 which is in fluid communication at its one or upstream end 5 with the receiving and

Mixing region 7 and at its other end to one upstream end of a second or intermediate region 11. The intermediate region 11 is in turn connected at its other end to one or upstream end of a third or downstream region 10. The other or downstream end 6 of the region 10 is in fluid communication with the collection region 8. The first, second and third capillary regions 9, 10, 11 all preferably have a substantially rectangular cross-section, as can best be seen from Fig. 5, and preferably extend parallel to one another, the capillary regions 9 and 11 or 11 and 16 and their ends being connected to one another via capillary segments lying sextant thereto.

The width dimension of the first and second capillary regions 9, 1Λ (i.e. the distance between the side walls of the reaction capillary when viewed along its longitudinal axis) in the present embodiment is essentially equal to and smaller than the width dimension of the downstream capillary region 10. It is to be understood, however, that the invention is not limited to the described cross-sectional configuration or width dimension of the reaction capillary. In the thickness direction, i.e. in the direction perpendicular to the plane of Fig. 1 (see the distance between the bodies 2 and 3 in Figs. 2-4), at least the upstream first and downstream third regions 9, 10 differ from one another. In particular, the thickness dimension of the downstream region 10 is greater than that of the upstream region 9. The intermediate region 11 may have a thickness dimension intermediate that of the upstream and downstream regions 9, 10. In the preferred embodiment, each location in the downstream capillary region has a cross-sectional area that is greater than or equal to that of another location located in the downstream capillary region with respect to the first-mentioned location.

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As a result of the different thickness dimensions of the capillary regions 9, 10 and 11, different capillary forces are generated therein under the assumption that the other capillary-effective influencing factors are otherwise the same, and in particular the capillary force in the upstream region 9 is greater than in the downstream region 10. The capillary forces generated in the intermediate region 11 can be adjusted by suitable selection of the thickness dimension of this region so that they are equal to or smaller than the capillary forces in the upstream region 9 and/or equal to or greater than in the downstream region 10.

As can be seen in particular from Fig. 2-4, the thickness dimension along the capillary regions 9, 10, 11 from their upstream and downstream ends is not constant. Rather, it preferably changes continuously, if desired also in steps, with the thickness dimension at the upstream ends of the capillary regions being smaller than at their downstream ends. Each capillary region 9, 10, 11 of the reaction capillary 4 preferably has a substantially conical or wedge-shaped cross-sectional configuration in the longitudinal direction (ie in the flow direction). In contrast, as shown, the thickness dimension of the collection region 8 at the downstream end 6 of the reaction capillary 4 can be constant. Ventilation holes for venting the collection region 8 are indicated by the reference numeral 12 in Fig. 1.

The different capillary forces caused by the different cross-sectional shape, in particular the thickness, along the reaction capillary 4 mean that the flow rate of the sample reagent mixture along the upstream capillary region 9 is greater than along the downstream capillary region 10, while transitional conditions can prevail along the intermediate capillary regions 11. The higher capillary forces in the upstream capillary region 9 have a strong, constant pulling effect on the

This results in the reagents located in the receiving and mixing area 7 being pumped into the reaction capillary 4, and the higher flow rate along this capillary area causes the sample-reagent mixture to be "pumped" into the subsequent capillary areas 10, 11, where there is less capillary action. This achieves and maintains a positive flow of the sample-reagent mixture along the entire reaction capillary 4 and into the collection area 8.

The dimensions of the reaction capillary 4 in the thickness direction from its upstream to its downstream end 5 or 6 must basically be adapted to the respective sample-reagent mixture to be examined. Depending on the mixture, the thickness dimension at the upstream end 5 can be between 1 and 100 µm and at the downstream end 6 between 5 and 500 µm. Preferably the reaction capillary is between 50 and 100 µm thick at the upstream end 5 and between 200 and 300 µm thick at the downstream end 6.

Other thickness dimensions can also be provided and the thickness dimension of the reaction capillary along its length need not always change from a region of lesser thickness to a region of greater thickness in the direction of flow. In a preferred embodiment, however, the cross-sectional area of the reaction capillary increases continuously and steadily (e.g. in the form of a linear increase) from the upstream to the downstream end and across each of the capillary regions 9, 11 and 10. Alternatively, if desired, intermediate regions of lower flow velocity can also be preceded by regions of greater flow velocity.

It should also be noted that the capillary effect or the flow velocity along the reaction capillary can also be influenced or changed by factors other than the cross-sectional design. Without claiming to be complete, these are in particular the

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Surface quality, particularly roughness of the capillary walls, the material from which the test element is made, particularly any additives or moisture contained therein, internal stresses and material orientations from the manufacture of the test element and the like.

Preferably, the test element is made up of injection molded parts made from a suitable transparent plastic material such as polycarbonate, polystyrene, polymethyl methacrylate or resins based on acrylonitrile-butadiene-styrene. Polymethyl methacrylate (PMMA) is particularly preferred because of its good optical properties, weldability and processability in the injection molding process. However, it is understood that, if desired, other materials, including inorganic materials such as glass, can also be used. It is also intended to use resins that contain antistatic materials as additives. To suppress the static charge, it is intended to use a plastic with carbon black as an additive.

When using polymethyl methacrylate (PMMA), one of the following additives can be used:

a) "Carbon Black Pigment" from Degussa, Hanau, Germany, with the trade name Masterbatch CB 51. This additive is added at a percentage of 3-5%.

b) An antistatic agent from ICI with the trade name ATMER 129, which contains glycerol monostearate (90%) and some glycerol disterate. This additive is added in percentages of e.g. 0.125%, 0.25% or 0.5%.

c) An antistatic agent manufactured by Lanko, U.K., with the trade name LANKOSTAT JP, which

Contains lauric acid diethanolamine. This additive is added, for example, at a percentage of 0.5%.

Furthermore, it is understood that instead of a reaction capillary 4 with capillary regions running essentially parallel to one another, a straight extension of the reaction capillary or any other configuration, e.g. a spiral arrangement, of the capillary regions can also be provided. The provision of an intermediate capillary region 11 is not obligatory. Within the scope of the invention, it is also provided that when the test element is used for a competitive detection method, the sample-reagent mixture contains an antibody, which is not necessary in a non-competitive detection method. In a non-competitive detection method, the antibody is contained in the body fluid (e.g.

in AIDS tests).

Using a test element of the type described above, an agglutination test can be carried out, for example, as follows. A certain amount of the liquid to be tested, e.g. a person's urine or blood, is successively applied to the receiving and mixing area 7 of the test element 1 together with certain amounts, e.g. one drop each, of a reagent containing antibodies, a buffer reagent and a reagent with latex particles coated with a substance being sought or a metabolite of the substance being sought and then gently mixed together in the receiving and mixing area 7. In a competitive detection method, the reagent also contains antibodies. Due to the strong capillary action in the upstream capillary area 9, the mixture is automatically drawn through the opening 5 into the reaction capillary 4 and "pumped" into the subsequent capillary areas 10 and 11.

An agglutination-active state of the sample-reagent mixture is maintained solely by its flow along the reaction capillary, so that no external forces are required on the test element to keep the mixture moving. The formation of visible agglutinations occurs essentially along the downstream capillary region 10 and can be visually observed and evaluated in particular at the collection region 8. As a result of the pumping effect of the upstream capillary region 9, the time that elapses for the flow of the sample-reagent mixture from the upstream to the downstream end 5 or 6 of the reaction capillary 4 or until the collection region 8 is completely filled is highly reproducible and can be optimized in particular for the respective sample-reagent mixture to achieve maximum sensitivity.

The test element is particularly suitable for detecting cocaine metabolites. For this purpose, a latex coated with benzoylecgonine, a monoclonal antibody against benzoylecgonine and urine as a body fluid are used. However, the invention is not limited to such use.

Example

A test element according to Fig. 1 was manufactured using a polymethyl methacrylate plastic (PMMA), e.g. that which can be obtained under the trade name Diacon, by injection molding and ultrasonic welding according to a process described in more detail below.

The dimensions of the reaction capillary were: 30

Length of capillary areas 9,11,10: approx. 50 mm each; Width of upstream and intermediate capillary areas 9,11: approx. 2 mm each; Width of downstream capillary area 10: 3 mm; Thickness dimension of capillary areas, measured at their upstream and

Downstream: upstream capillary region 9: 80 or pi, intermediate capillary region 11: 130 or 170 um, downstream capillary region 10: 200 or 240 um; thickness of the collection region: 240 um.

A sample reagent mixture of the type mentioned above for the detection of narcotic substances in urine was applied to the receiving and mixing area 7 of the test element and the time was measured that elapsed for the sample reagent mixture to move along the individual capillary areas until the collection area 8 was completely filled. The following average times were determined: 12 seconds for the upstream capillary area 9, 26 seconds for the intermediate area 11 and 140 seconds for the downstream capillary area 10 including the collection area 8. Overall, the agglutination reaction took about 3 minutes, which is considered to be optimal for the reagent mixture in question in terms of evaluation sensitivity. The coefficient of variation of the response time ([standard deviation/mean] × 100) for any set of measurements should preferably not be more than + 10 %.

A preferred method for connecting the plate-shaped bodies 2, 3 produced by injection molding a plastic material, in particular PMMA, is described below with reference to Fig. 6A, 6B. Fig. 6A and 6B show the plate-shaped bodies 2, 3 in the state before and after joining by ultrasonic welding. As described at the beginning, a continuous (or interrupted if desired) web 13 extends circumferentially along the areas 7 and 8, but at least along the reaction capillary 4.

The web 13 has a substantially rectangular or slightly trapezoidal cross-section in the initial position according to Fig. 6Aacgr;. The web 13 on the upper plate-shaped body 3 corresponds to a complementary groove 15 in the lower plate-shaped body 2 with a cross-sectional configuration such that the web 13/ is only partially received therein, as shown.

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In particular, the groove 15 has a substantially frustoconical cross-section tapering inwards, so that the lateral edges 14 of the web 13 can engage with an intermediate point of the conical side walls 16 of the groove 15. If, in this initial position, ultrasonic energy is applied to the plate-shaped bodies 2, 3 in a manner known to those skilled in the art, the areas of the web 13 that engage with the side walls 16 of the groove 15 melt, as is indicated in Fig. SB at 17.

Under the welding pressure exerted on the plate-shaped body 2, 3, the web 13 will therefore move further into the groove 15 until the front surface of the web 13 comes into contact with the bottom surface of the groove 15. At this stage, not only is the thickness of the reaction capillary 4 and the collector region 8 located between the adjacent web regions fixed, but these regions are also sealed fluid-tight to the outside and a firm connection of the plate-shaped bodies 2, 3 is created. The different thickness of the reaction capillary 4 along its longitudinal extent can be taken into account either by appropriate shaping, in particular the depth of the surface sections of the upper and/or lower plate-shaped body forming the reaction capillary. As an alternative to this, said surface sections can also be sprayed with a constant depth, while during welding the plate-shaped bodies 2, 3 are held in an oblique initial relationship to one another (not shown). This results in one part, in Fig. 1 the left-hand part, of the web 13, taking up a different position in the corresponding groove 15 than the other right-hand part of the web. If, under these circumstances, ultrasonic energy is applied to the plate-shaped bodies 2, 3, the left-hand web part will sink deeper into the groove 15 under the welding pressure than the right-hand web part, with a correspondingly different cross-sectional formation of the

reaction capillary 4. It is understood that the arrangement of the welding web and the welding groove can also be reversed, with the former being formed on the lower and the latter on the upper plate-shaped body.

The invention has been described above using a preferred embodiment. It is understood that the invention is not limited to this. In particular, the inventive principle of forming an agglutination capillary can also be applied to test devices of any other type. Furthermore, the different capillary action of the upstream and downstream areas of the reaction capillary can be achieved not only by different thicknesses, but generally by different cross-sectional dimensions of the capillary areas. The term "reaction capillary" also includes no or only very slight capillary action in relation to the downstream area.

Claims (13)

Protection claims 1. Test element for agglutination tests with at least one reaction capillary (4) through which a sample-reagent mixture can flow, which is connected on the inlet side to a receiving and mixing area (7) for the sample and the reagents, whereby the sample and the reagents form a sample-reagent mixture, characterized in that the reaction capillary (4) comprises an upstream area (9) with a capillary effect for creating a higher flow rate for the sample-reagent mixture than along a downstream area (10, 11) of the reaction capillary. 2. Test element according to claim 1, characterized in that the upstream and downstream regions (9, 10, 11) of the reaction capillary (4) merge into one another. 3. Test element according to claim 2, characterized in that the transition between the upstream and downstream areas is continuous. 4. Test element according to claim 2, characterized in that the transition between the upstream and downstream areas is step-like. 5. Test element according to one of the preceding claims with at least one pair of plate-shaped bodies (2, 3), between which a reaction capillary (4) with a substantially rectangular cross-sectional configuration is formed, eo oesosoaoi he^, characterized in that the dimension of the reaction capillary (4) in the thickness direction along the upstream region (9) is smaller than along the downstream region (10, 11). •&ggr; 19 ~ ·» · • · Il f I « 6. Test element according to claim 5, characterized in that the reaction capillary (4) widens in the thickness direction from the upstream to the downstream region. 7. Test element according to claim 6, characterized in that the expansion of the reaction capillary (4) occurs continuously. 8. Test element according to claim 6, characterized in that the expansion of the reaction capillary (4) takes place in stages. 9. Test element according to one of claims 5 to 6, characterized in that the dimension of the reaction capillary (4) in the thickness direction in the upstream region is between 1 and 100 µm and in the downstream region between 5 and 500 µm. 10. Test element according to one of claims 5 to 9, characterized in that the dimension of the reaction capillary (4) in the thickness direction in the upstream region is between 50 and 100 µm and in the downstream region between 200 and 300 µm. 11. Test element according to one of the preceding claims, characterized in that the reaction capillary (4) has a substantially S-shaped course and contains an intermediate region (11) between the upstream and downstream regions (9, 10), the capillary action of which creates a flow rate for the sample-reagent mixture that is equal to or less than that along the upstream and/or equal to or greater than that along the downstream region. 12. Test element according to one of the preceding claims, characterized in that it contains a collection area (8) for receiving the sample-reagent mixture after it has passed through the reaction capillary (4). 13. Test element according to one of the preceding claims / characterized in that it is composed of injection-molded parts that are made of a transparent plastic material.