HRP940755A2 - Diagnostic device in the form of a sheet - Google Patents

Description

The invention relates to a solid diagnostic tool consisting of several functional fields, which is used for proving and for the quantitative determination of substances or analytes in biological fluids. The invention further relates to the method of using this agent, where upon contact of the agent with the liquid, the analytes react with specific biologically related binding partners and are demonstrated by means of labeling reagents.

In diagnostics, it is necessary to identify and determine specific compounds, control drug data, quantify physiologically active compounds or products that follow from them, and enable the diagnosis of infections. Here, the methods of immunoanalysis (RIA, ELISA, agglutination test) are of particular importance. Specific binding reactions, which are used in testing, are not limited to immunological interactions, such as interactions between antigens, i.e. haptens and antibodies, but also biologically related interactions, such as lecithin-sugar, active substance-receptor.

Although the existing tests are sensitive and specific, however, due to the long duration of the test (usually several hours or even days) and the numerous stages of the test, such as immuno-reactions, washings, enzymatic reactions, they do not represent any convenient forms for application. Long test times incompatible with use in emergency diagnostics.

Test elements integrated with dry chemistry, such as those described in the proposed invention, simplify test performance and shorten test times.

No layered assay element has been described in which all immunoreaction components of a heterogeneous immunoassay are integrated with solid-phase detection as well as unfolding of function and "bound-free" disassembly.

While in the performance of test strips, the immunoreaction phase and separation of the bound and free phase, in heterogeneous testing, is carried out with a directed flow of liquid, in the performance of test elements that work with thin layers, laminated one above the other (film technology), the possible forces are controlled by diffusion, procedures aimed at decreasing concentration. DOS 33 29 728 (JP P144 341/82) and EPA 0 097 952 (JP 114 359/82) work with fluorescent marking. The marker has a very small molecular weight and thereby accelerates water diffusion processes. In addition, the test must be carried out at an elevated temperature. In the first of these two cases, both the free and the bound phase are read. In film technology, solvent extraction is enhanced either by hydration of the swellable component or by filling the capillary cavity. In versions with layers laminated one above the other, only the highest and lowest layers are available without major detection difficulties.

At the end of the reaction phase, it is difficult to react the reagents with the components located between the layers. When constructing test strips with zones located one behind the other, as used in the invention proposed here, in principle each zone, both from above and also from below, is easily accessible for measurement, and also for adding any necessary reagents.

The subject of the invention is a layered diagnostic tool that contains all reagent components integrated, which contains not only all the components necessary for the performance of the function, but also the performance of the function itself, with which an analyte with biologically related properties can be demonstrated, whereby the analyte solution is brought into contact with the functional area of the agent provided for it, and the analyte can be demonstrated in a single functional area, in the solid phase zone, via the system that produces the signals.

Other or further analytes, as constituents of the same solution, can be detected simultaneously with the agent, if they. have biological affinity properties different from the first. They will also prove themselves in a single functional area, a self-contained solid zone, in the same way as the first analyte. The functional area for the detection of other and further analytes is located in the layered means in front of or behind the functional area for the detection of the first analyte. The device can also contain multiple zones of the solid phase, which are available for analytes and different measurement areas of these analytes. The agent and its ingredients are primarily present in dry form.

The layered diagnostic means consists of one or several strips, arranged one behind the other, of materials that absorb aqueous solutions. The strips are fixed on a solid base. They contain the ingredients of the reagents required for the diagnostic tool in question and thus become functional fields or functional areas. The functional field (solvent application zone), located at one end of the strip means, comes into contact with the analyte solution by immersion in the analyte solution or by application of the analyte solution. The solution passes through all functional areas. The ability to absorb the carrier materials, from which the tapes are made, causes a flow of liquid that stops at the other end of the tape means. The analyte can also be placed in the middle region of the device and finally a flow of liquid is produced from one end of the device to the other.

In this case, the sample does not have to be placed directly on the part of the test strip that is chromatographed. It can also be placed on the absorbent material lying on the test strips and has the function of removing blood cells from the sample.

Then the sample, after filtration, comes to the test strip. During this filtration, the addition of reagents can be carried out at the same time by releasing the dry component that is in the filter. With the help of such components, disturbing factors can be removed from the solution. Thus, possibly with a suitable oxidizing agent, the ascorbic acid contained in the sample, which interferes with the use of oxidase and peroxidase as a marking agent, can be rendered harmless. Furthermore, the filter can also have the function of an absorber, which removes disturbing factors from the sample by absorption. The rinsing agent application zone, or the zone located behind it, can also take on the filtration, absorption and reagent mixing functions for conditioning the test sample. Solvent distribution. in individual functional areas, it depends on the absorption capacity and dimensioning of the materials used.

The solvent application zone can function as a volume metering element, as described in DE 30 43 608, DE 23 32 760, USP 3, 464, 560, USP 3, 600, 306, USP 3, 667, 607, USP 3, 902, 847, USP 4, 144, 306 and USP 4, 258, 001. It may contain various reagents in dry form necessary for the functions of the test element. The solvent application zone can be a piece of thin absorbent paper located at one end of the test element which, by simply dipping it into the solution, eg the sample. or with a short splash of running water, it absorbs a defined volume of liquid and finally delivers the liquid to the following zones more slowly and in a controlled manner. The solvent application zone is dimensioned to provide enough liquid for it to travel to the other end of the agent, to the end of the absorption zone.

Between the solvent application zone and the absorption zone there are functional areas in which the reaction ingredients for the test are located and in which all reaction phases of the test run take place. Part of the reaction ingredients for conducting the test can also be placed in the sample application zone. The absorption zone has the task of taking up excess reagents and reaction products of the system, which move freely and produce signals.

Carrier materials capable of being absorbed in the form of one or more strips as constituent parts of different functional areas can optionally be made from cellulose, from chemically derivatized cellulose or from plastic with a porous or fibrous structure with sufficient hydrophilic properties, as well as from particles immersed in a polymer membrane, such as cellulose or silica gel and further from hydrophilic but not water-soluble natural processed products. A combination of strips consisting of different materials can be used. The choice of suitable absorbent materials depends on the requirements of the respective diagnostic tool.

In various embodiments, the biologically related components of the reagents are immunochemical, diagnostic agents, antigens, haptens or antibodies. In the case of proving glucoproteins or oligosaccharides, which bind to lectins, the biologically related reaction partner can be a specific lectin, the other biologically related reaction partner can be an antibody, which is directed to the binding site of the analyte different from the lectin. In the case of proving microbial active substances, one of the reaction partners can be a receptor for the active substance, and the other binding partner can be an antibody directed towards the second binding site of the active substance.

The biologically related binding partner is during the course of the reaction or already connected to the support material in the functional area intended for the detection of the analyte (solid phase zone). It is also called the binding partner of the solid zone. One or more remaining binding partners are contained in the carrier materials. They are marked.

Of the various known labeling options, preference is given to enzymatic labeling. This requires chromogenic substrate systems that produce fluorescence or luminescence. Chemiluminescent labeling is a further example of labeling, which is measured only after the addition of the reagent. In doing so, the chemiluminescence or fluorescence itself, which is excited by it, can be measured. Fluorescence labeling is most often measured, and no reagents need to be added. However, also as with the use of certain rare earth chelates, it may be desirable to produce the measured fluorophore only by adding a reagent or to add a second fluorophore, which is excited with the first or excites the first. Fluorescence can be measured simply with time resolution or as fluorescence polarization.

One reagent, required for proof, can be brought into reaction with the immunocomplex to be proved in a separate process in different ways. Part of the system that produces the signals may be in the solid phase zone. The labeling reagent, required for proof, can be added later, after sufficient washing of the solid phase in the heterogeneous immunoassay with detection in the binding phase in various embodiments. The possibilities are, for example, the following.

The introduction of regenase included parallel to the main flow of liquid, the slower flow of liquid coming out of the reservoir of the rinse aid: and before the zone it joins the marked component. A liquid stream connected in parallel can be guided by the use of a slower absorbent capable of being chromatographed, possibly suitable chromatographic paper or paper impregnated in places with blocking components that "cut the path", as for example in the discharge of polymers (e.g. polyvinyl alcohol, dextran). which gives high viscosity.

Application of the reagent can be performed after sufficient washing of the solid phase (= end of chromatography) using a low pressure element which is a solid component of the test element. "Low pressure" can be produced mechanically, or by removing spacers, by the action of a fluid stream. Mechanical low pressure can be exerted, for example, by one of the elements contained in the reagent by low pressure of the cover or paper held by a spacer holder. .Removal of the element contained in the reagent container with the action of the liquid current can be achieved, for example, by mutual lamination of the solid phase, the polymer soluble introductions and the reagent carrier (e.g. suitably impregnated paper).

Delayed introduction of the reagent into the liquid stream can be done by using a microencapsulated reagent, which only comes out of the capsule after sufficient washing of the solid phase, or by entraining the reagent captured in a matrix with a slowly dissolving component.

One possibility for the special case of enzymatic labeling looks as follows. When using the peoxidase marker, the glucose oxidase zone can be placed in front of the solid zone. Glucose and also a chromogen will then be incorporated into the liquid stream, which can lead to staining behind the glucose oxidase. Significant coloration will be observed when sufficient H2O2 is produced by the oxidase at a suitably high concentration of peoxidase. This formation of peoxide continues slowly, reaching an optimal concentration and finally a high concentration that leads to the inhibition of the enzyme and thus automatically stops the formation of coloration. This coloring can be modified if a scavenger - H2O2, eg thioether, as a mild reducing agent or catalase enzyme - is incorporated into the oxidase zone, or in front of it.

In this example, delayed inclusion, with the help of enzymes, produces a reagent for proving the labeling agent. Color formation in the solid phase zone occurs only after the non-specifically bound marker is sufficiently washed away from that zone with the liquid stream.

There are several possibilities for the preparation of the solid phase zone. The components fixed there can be chemically covalently or absorptively bound to an absorbable support, which is part of the test element. These components can also be connected to the dispersion of particles, which, after being applied to a suitable carrier, remain fixed at the place of application. For example, cell suspensions that carry specific receptors on the surface, such as possibly Staphylococcus aureus Cowan I cells or latex particles, that carry a biologically related binding partner on the surface, fixed in a paper matrix, are suitable. The components bound on the pipettable carrier, as well as the unbound components of the test strip, can be dried on the absorbent matrix of the element by air drying; a freeze-drying process is not necessarily necessary.

As embodiments, for example, several test flows will be presented, which should be viewed independently of the marker used. For simplicity, they are described only for the detection of a single analyte with a diagnostic tool.

In the event that the analyte has only one single biologically related binding site or only one of several biologically related binding sites is used, the following two forms of performance will be presented, which in principle correspond to the competitive immunoassay.

The solid phase binding partner is covalently or absorptively linked to the support material of the solid phase functional region. The analyte solution is made mobile by a predetermined amount of labeled analyte contained in the diagnostic agent. Both components travel to the functional field, which contains the binding partner in the solid phase, and compete for binding with the binding partner in the solid phase. If the proportion of analyte is high, compared to labeled analytes, less labeled analyte will bind. If it is low, a lot of labeled analyte will bind.

The binding partner from the solid phase is in the functional area and is placed as an unbound component in front of the functional area of the solid phase. The incoming solvent front transports it into the functional region of the solid phase. This binding in the solid phase is achieved using a biologically related binding system, which is independent of the analyte binding system. The biotin-conjugated binding partner binds to the carrier-bound avidin. Immunoglobulin, such as IgG, as a binding partner, will be fixed via its Fc part on the carrier to bind protein A from S. aureus or. carrier-bound, non-idiotypic antibody.

The analyte and labeled analyte compete as constituents of the diagnostic agent during the unfolding function for binding to the solid phase binding partner, as previously described. This competition reaction takes place partly with the dissolved and partly with the binding partner of the solid phase already bound by the solid phase.

If there are two binding sites of different specificity in the analyte, one can imagine several forms of execution of the diagnostic agent, which in principle correspond to the sandwich immunoassay. Two of these embodiments will also be shown below.

If the binding partner in the solid phase is covalently or absorptively linked to the carrier material of the functional area of the solid phase, the analyte forms a binary complex with the labeled binding partner, which enters the functional area of the solid phase with the solvent and reacts there with the binding partner from the solid phase, whereby forms a ternary complex bound to the solid phase, which can be demonstrated by the marker of the first binding partner. The excess labeled binding partner separates with the solvent into the next functional area, the absorption zone.

If the binding partner in the solid phase of the diagnostic agent is not present in a bound form and is moved by the solvent, both biologically related reaction partners of the analyte are brought into the functional region so that the analyte reacts with both partners simultaneously or sequentially and the resulting ternary complex ultimately travels to the functional region. the solid phase region, where, as described earlier, it binds independently of the analyte to the solid phase of a biologically related system.

To illustrate the previously described and further embodiments, which correspond to the immunometric test principle, the principle of indirect antibody detection or the ELA principle (enzyme-labelled antigen, e. Enzyme-Labelled-Antigen) of immunoanalysis; the distribution of the components of the agent in the functional areas as well as the development of the reaction of the composition of the solid phase complex, the amount of which is a measure of the concentration of the analyte in the sample, are presented in reviews I and II.

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It was found that one fully integrated test strip, which works according to the principle of heterogeneous immunoanalysis with detection in solid phase, is not only feasible, but can be read within a time period of less than one hour, while the possibility of quantification and sensitivity of conventional RIAs or ELISAs is reached. It was possible to prove traces of components in the range of 10-16 mol/1 with a required sample amount of 10-16 mol, which corresponds to, for example, approx. 1 pg, with a reaction time of less than 30 minutes, at room temperature. However, tests with lower sensitivity requirements can also be performed with the described arrangement. Standard curves were obtained over two to three decades when reading with a Sanoquell reflectometer (Quelle company). Chromatography time for the test element, including gradual color development, is a maximum of 16 minutes. The reading can also be done visually. With HCG as an analyte, in the case of glucose oxidase bound on the solid phase and with a peroxidase marker, the beginning was in the measurement range at 0.3 ng/ml (corresponding to 3 U/1).

In the following examples, as a concrete form of implementation, the application of the principle of competitive double antibody testing is presented. In this test configuration for the determination reaction and the separation procedure, four components were brought into the reaction, one after the other, where the critical values are the reaction time and the concentration of the reactants. The example should not be considered limiting in any way, but only serves to better explain the subject of the invention.

Examples

Fully integrated enzymatic immunochemical test strips for proving HCG with a built-in chromogenic substrate system.

1.1 Reagents

1.1.1 HCG and peroxidase conjugate

HCG with a specific activity of approx. 3000 U/mg was obtained from Organon. Horseradish peroxidase was purchased from Boehringer, Mannheim (catalog number 413 470). The heterobifunctional reagent N-γ-maleimido-butyryloxysuccinimide (GMBS) was purchased from Behring Diagnostics and, as described by Tanimore et al., 1983, in J. Imm. Meth. 62, 123-131, was chemically converted with HCG. 2-iminothiol-hydrochloride (Sigma, Cat. No. I 6256), described by King et al., 1978, in Biochemistry 17, 1499-1506, was chemically converted with peroxidase. A conjugate was produced from GMBS-HCG and iminothiol-peroxidase, as described by Tanimori et al. The crude conjugate was purified by gel chromatography on Utrogel ACA 44 (LKB company). The fraction in which approximately 1-2 peroxidase molecules per HCG molecule were bound was used for testing. The conjugate was diluted with Enzignost IGE, an incubation medium from Behringwerke, Best. o. OS D, which is referred to below as incubation medium for short.

1.1.2 Antibodies

Antibodies against HCG were obtained by immunization of guinea pigs, and antibodies against guinea pig IgG were obtained by immunization of goats. IgG fractions were obtained from serum by precipitation with ammonium sulfate and chromatography with anion exchange and further purified by immunoabsorption. The applied methods are described in the book "Immunologische Arbeitsmethoden", Helmut Friemel, publisher Gustav Fischer Verlag, 1984, Stuttgart. Anti-HCG antibodies were diluted in the above conjugate dilution buffer.

1.1.3 Glucose oxidase

Glucose oxidase was obtained from Aspergillus niger as a solution with 300 U/mg (company Serva, catalog number 22737) Oxidase. glucose is extremely diluted with the incubation medium.

1.1.4 Glucose, tetramethylbenziidine

α-D-glucose and tetramethylbenziidine hydrochloride were purchased from Serva, catalog number 22720 and 35926, respectively.

1.2 Preparation of funds

The layered functional area was prepared as follows.

To prepare the rinse aid application zone, a thin sponge fabric from the company Kalle, which is a dry-pressed synthetic sponge made of regenerated cellulose, was cut to a size of 20 x 6 mm. It was soaked with a solution of 50 mg of glucose and 0.75 mg of tetramethylbenzlidine hydrochloride per ml of water and dried with air current.

Anti-HCG antibody conjugate and glucose oxidase (5 μl each with 25 μ/ml, 100 μl/ml, and 0.1 mg/ml, respectively) were applied at regular intervals on a piece of paper NM no. 1 of size 45 x 5 mm (Macherey & Nagel) for each other and air dried.

As a solid phase zone, a piece of paper no. 597 from Schleicher & Schüll, size 5 x 5 mm, covalently coated with anti-guinea pig IgG antibodies. To this, antibodies were bound to cyanogen bromide-activated paper as described by Clarke et al., 1979, Meth. Enzymology, Vol. 68, 441-442.

A piece of paper no. was used as the absorption zone. 2668/8 (by Schleicher & Schüll), size 20 x 5 mm.

Four papers of 0.5 - 1 mm were fixed on a solid base, so that they were placed on top of each other using double-sided adhesive tape (Tesaband by Beiersdorf), and thus a test strip with a width of 5 mm was obtained.

1.3 Implementation of the test

To perform the test, 200 μl of diluted HCG was applied to the incubation medium on thin paper.

l.4 Result

Chromatographic development of test elements. and automatic color development were concluded after 15 minutes at room temperature and could be read visually or with a reflectometer.

When reading the solid phase zone (Paper No. 597) with the Quelle Sanogel blood glucose meter, the following results were obtained.

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The following results were obtained with the urine reading device, when tested with Behringwerke's Rapimat strips, with the same test strips.

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The construction of the test strips shown here can also be realized if the glucose oxidase and anti-HCG antibodies are in the same zone. The corresponding shorter test strip then has a test time of approx. 10 minutes.

Claims (13)

1. Analytical means for proving or determining the component of a biologically related binding pair (analyte) in a liquid, which consists of several layered zones arranged one behind the other, and over their edges they touch each other absorbently, which means contains a zone for applying a rinsing agent (EMAZ) at one end and the absorption zone (SZ) at the other end of the means, - as well as further absorption zones that are located between these two, in which the reaction participants capable of interacting with biologically related analytes are arranged so that the reaction participants are which can interact with each other placed spatially apart, indicated by that a) the reactant is covalently or absorptively or through a biologically related interaction fixed in the zone, the solid phase zone (FPZ), which is located between EMAZ and SZ and is in contact with the SZ, or is covalently or absorptively or via a reaction that takes place in the medium of biologically related mutual action connected with further rectants fixed in FPZ, b) further labeled reactant (conjugate) is located unconnected in the zone between EMAZ and FPZ i c) the analyte application zone is EMAZ or the zone between EMAZ and SZ. 2. A layered diagnostic tool according to claim 1 for the detection of two or more analytes with one or more biologically related binding sites in the solution, characterized by the fact that, depending on the analyte, it contains a spatially separated zone of the solid phase, which is equipped with specific binding partners for the analytes in question , connected by a carrier, and in which the analytes are proved separately. 3. Means according to claim 1 or 2, characterized by the fact that EMAZ has the function of an element for volume dosing and delivers at least so much liquid to the next zone that the liquid reaches the end of the NW by capillary forces. 4. The agent according to one of the claims 1 to 3, characterized by the fact that the EMAZ is a plastic sponge or a special layer consisting of hydrophilic polymers, which may contain chemicals, buffers or other substances necessary for testing, if necessary. 5. Means according to one of claims 1 to 4, characterized in that the sample application zone retains blood cells. 6. Means according to one of claims 1 to 5, characterized in that the sample application zone is laminated on one of the layered zones of the part of the chromatography means and is in absorbent contact with it. 7. The agent according to one of the claims 1 to 6, characterized by the fact that all or a part of the reactants required for proving labeling are located in one or more layered zones of the agent, or are laminated on one of the layered zones of the chromatographic part of the agent, and stand with it in an absorbing touch. 8. A method using an agent according to one of claims 1 to 7, characterized in that the reactants are present in the agent in a dehydrated form and are rehydrated or solvated with liquids introduced into the agent. 9. The method according to claim 8, with the use of an agent according to one of claims 1 to 7, characterized in that after placing a liquid sample, which contains analytes, on the EMAZ, or after placing the sample on the sample placement zone and placing the rinsing agent on the EMAZ , the water fluid reaches the end of the SZ by capillary forces and thus a reaction is established between the reaction participants, contained in the agent, and the analyte, and after the chromatographic separation of the marker that is not specifically bound on the solid phase, the amount of marker in the solid phase zone is determined, which is a measure for the concentration of the analyte in the sample. 10. The method according to claim 8 or 9, with the use of an agent according to one of claims 1 to 7, characterized in that the reactions taking place in the agent are basically based on immunological reactions demonstrating competitive immunometric or sandwich-immuno tests or indirect evidence of antibodies with labeled antibodies or by proving antibodies with a labeled antigen. 11. The method according to one of claims 8, 9 or 10 with the use of an agent according to one of claims 1 to 7, characterized in that the labeling agent is a Fluorophor, which is proven or measured directly, or is proven or measured after the addition of a reagent present in agent, or by the addition of a reagent present in the agent, a Fluorophore is formed from the marker, which is proven or measured directly or after the addition of a further reagent. 12. The method according to one of claims 8 to 10, with the use of an agent according to one of claims 1 to 7, characterized in that the marking agent is a compound that causes chemiluminescence, wherein chemiluminescence can be demonstrated or measured after the addition of the agent present in the reagent. 13. The method according to claims 8, 9 or 10, with the use of an agent according to one of claims 1 to 7, characterized in that the labeling agent is an enzyme whose activity can be determined with the help of a reagent present in the agent.