JP2504923C - - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunological measurement method using a sheet-like analysis device. 2. Description of the Related Art In a diagnostic method, a specific compound can be identified and measured, so that it is possible to administer a drug, monitor a biologically active compound or a secondary product thereof, and diagnose an infectious disease. It has become. In this regard, immunoassays (RIA, ELI
SA and agglutination tests) are of particular importance. The specific binding reactions used in these tests are not limited to immunological interactions, such as antigen-antibody or hapten-antibody interactions, but also interactions with biological affinity, such as lectin-sugar or active compound-receptor. The body is also used. [0003] Although conventional tests are sensitive and specific, they require a long time to test (often hours or even days) and many test steps such as Since it requires an immune reaction, a washing step, and an enzymatic reaction, it does not exhibit a convenient application form. The long test times mentioned above are not suitable for use in emergency diagnosis. An integrated dry chemistry test element as described in the present invention simplifies test performance and reduces test time. Immune reaction, performance of action and "binding phase-" of heterogeneous immunoassay using solid phase detection.
Sheet-like test elements in which all components for "bound-free" separation have been integrated have not been described at all. In test strip assemblies, the immunoreaction step, and the separation of the bound and free phases, is carried out in a heterogeneous test with a directed liquid stream, whereas the thin layers are superposed on one another. In the test element assembly (film method), control by diffusion and a process directed by a density gradient is a propulsive force.
The fluorescent label is disclosed in West German Patent Application Publication No. 3,329,728 (JP-A-57-144341).
No.) and EP A 0,097,952 (JP-A-57-114359). The label has a low molecular weight, thus facilitating a process that is controlled by diffusion. However, the test must be performed at an elevated temperature. In the first of these two cases, both the free and bonded phases were evaluated. In the film method, the absorption of the solvent takes place by hydrating the swellable component or filling the capillary voids. In the case of assemblies having layers stacked on top of each other, only the top and bottom layers are available for detection without major difficulties. After the reaction step has been performed, it is difficult to react the reagents with the components of the intermediate layer. In a test strip assembly having successive zones as used in the present invention, basically each zone is used for both measurement and addition of reagents that may be required. Can be approached from below or from below. [0005] The present invention comprises all reagent components and includes in an integrated manner the action sequence itself as well as all the components necessary for the action sequence; and Contacting the solution of the analyte with the working area of a device designed for this purpose and detecting the analyte at the single working area (solid phase zone) via a signal generating system allows for biological affinity. The invention relates to a method for detecting or measuring an analyte having sexual properties. A second analyte or a further analyte as a component in the same solution is simultaneously detected by the device if these analytes have different biological affinity properties than the first analyte. can do. They are also detected in a single working area (the solid phase zone suitable for them) in a manner similar to the first analyte. The working area for the detection of the second or another analyte is located before or after the working area for the detection of the first analyte in the sheet-like device. The device can also include a solid phase zone that is appropriate for an analyte and various measurement ranges of the analyte. This device contains all reaction components and reagents in dehydrated form. [0006] The sheet-shaped diagnostic device consists of one strip or a plurality of strips of material having the ability to absorb aqueous solution. The strips are fixed to a solid support. They contain the necessary reagent components for a particular diagnostic agent, and thus constitute an active sector or area. The working sector (mobile phase application zone) located at one end of the strip-shaped device is brought into contact with the analyte solution by immersion or by applying the solution. The solution moves through all working areas. Liquid flow caused by the absorption capacity of the support material that makes up the strip stops at the other end of the strip-shaped device. The analyte can also be applied to a central region of the device, which can then induce a flow of liquid from one end of the device to the other. There is no need to apply the sample directly to the chromatography section of the device.
It can also be applied to absorbent materials located on the device and having the function of removing blood cells from the sample. The sample is suitable for the device after being filtered. In this filtration step, the addition of the reagent can be performed simultaneously by eluting the latter in a dry state from the components present in the filter. Such components can remove interfering factors from solution. That is, for example, ascorbic acid present in a sample, which interferes when oxidase or peroxidase is used as a labeling agent, can be rendered harmless by a suitable oxidizing agent. Furthermore, the filter may also have the function of an adsorbent to remove interference factors from the analyte by adsorption. Filtration, adsorption and reagent mixing functions to prepare the analyte for this test can also be performed by the mobile phase application zone. [0008] The solvent distribution in the individual working areas depends on the adsorption capacity and dimensions of the materials used. The mobile phase application zone may have the function of a metering element (German Patents 3,043,608 and 2,332,760, and U.S. Pat. Nos. 3,464,560, 3,
No. 600,306, No. 3,667,607, No. 3,902,847, No. 4,144
, 306 and 4,258,001). It can contain various reagents required for the function of the test element in a dry state. The mobile phase application zone is located at one end of the reagent element and is to be completely saturated with a predetermined amount of liquid by simply immersing or applying a solution of the sample, for example a sample, and then It may be a piece of fabric paper that releases the liquid more slowly and in a controlled manner than the subsequent zone. The mobile phase application zone is dimensioned to absorb enough liquid to move to the other end of the device, the end of the absorption zone. [0009] Between the mobile phase application zone and the absorption zone, the reaction components for carrying out the test are contained and there is arranged an active area in which all the reaction steps of the test run take place. Some of the reaction components for conducting the test may be stored in the sample application zone.
The absorption zone has the function of absorbing excess and freely movable reagent components and reaction products of the signal generating system. The absorbent support in the form of one or more strips as a component of the various working areas may, depending on the choice, be cellulose, a chemical derivative of cellulose, or a porous or fibrous structure and sufficient hydrophilicity. It may be composed of plastics having lipophilic properties, or particles such as cellulose or silica embedded in a synthetic membrane and hydrophilic but water-insoluble natural products. Combinations of strips composed of different materials can also be used. Suitable absorbent materials have been selected based on the requirements set for the particular diagnostic device. [0010] Reactive components having immunological binding properties, such as antigens, haptens or antibodies, can be incorporated into various aspects of the device. When detecting a glycoprotein or oligosaccharide that binds to a lectin, one reaction component having a biological affinity may be a specific lectin, but a second reaction component having an immunological affinity May be an antibody to a binding point of the lectin on another analyte. In the case of detecting an active compound of a microorganism, one binding partner can be a receptor substance for the active compound, while the second binding partner is an antibody to another binding point on the active compound. Can be. One binding partner with biological affinity becomes bound during the course of the reaction or is a support for an active region (solid phase zone) designed for the detection of an analyte. Already bonded to the material. It is also called a unique binding partner. Other binding partner (s) are present in the support. They are marked. Although various possibilities are known for the label, an enzyme label is preferable among them. It requires a chromogenic substrate system or a substrate system that produces fluorescence or chemiluminescence. Chemiluminescent labels are another example of labels that are measured only after the addition of a reagent. Either chemiluminescence itself or fluorescence excited by the latter (substrate system) can be measured. In most cases, fluorescent labels can be measured without the need for reagent addition. However, such as when certain rare earth element chelates are used, the addition of reagents results in the formation of a fluorophore that is to be measured only, or the excitation of the first fluorophore or the first fluorophore It may also be desirable to add a second fluorophore that excites Fluorescence can be measured at some point as a function of time or as fluorescence polarization. After the separation step, the reagents required for the detection of the immune complex (
(Crebex). Part of the signal generation system can be located in the solid phase zone. After thorough washing of the solid phase, the reagents required for the detection of the label can be released in a variety of ways at a slow rate in heterogeneous immunoassays involving detection of the binding phase. The following are possible examples: [0012] Application of the reagent by a liquid stream arranged parallel to the main liquid stream, but flowing at a lower speed and starting from the mobile phase reservoir and entering the zone containing the labeled component. This parallel liquid stream is transferred to a slower absorbing absorbent medium, such as a moderately slower chromatographic paper, or sometimes "components temporarily blocking the way", such as a solution. This can be controlled by using paper impregnated with a polymer (for example, polyvinyl alcohol or dextran) that imparts a high viscosity. After thorough washing of the solid phase (ie, after completion of chromatography), application of the reagent can be performed by pressing down the element that is a solid component of the reagent element. This "pulling down" can be done mechanically or by removing the spacing pieces under the action of the liquid flow. For example, depressing the reagent-containing element can be performed by depressing a flap or a piece of paper supported by a spacing strip. Downward movement of the reagent-containing element by the action of a fluid stream can be effected, for example, by laminating the solid phase, the water-soluble polymer and the reagent carrier (for example, a suitably impregnated piece of paper) with one another. [0014] Delayed introduction of the reagent into the liquid stream can be achieved by using microencapsulated reagents that emerge from the capsule only after the solid phase has been thoroughly washed, or by coating reagents attached to components that slowly dissolve in the matrix. Can be performed. One possible means provided in the case of a special enzyme label is as follows. That is, if a peroxidase label is used, the glucose oxidase zone can precede the solid phase zone. The glucose and chromogen can then be incorporated into the liquid stream, so that the color can be developed behind glucose oxidase. Recognizable coloration is only observed when sufficient H ₂ O ₂ is formed by the oxidase at moderate peroxidase concentrations. This peroxide formation starts slowly, reaches an optimal concentration, and eventually reaches a high concentration that results in enzyme inhibition and thus automatically stops color development. This coloration can be adjusted by incorporating an H ₂ O ₂ receptor, such as a thioether as a mild reducing agent, or the enzyme catalase into the oxidase zone or in front of the latter. In this example, the label detection reagent is produced by a delay circuit using an enzyme. Color development in the solid phase zone starts only after this zone has been washed sufficiently by the liquid stream to be free of non-specifically bound labels. Several means for preparing the solid phase zone are possible. The components immobilized thereon can be attached to the absorbent support that is part of the test element by covalent chemical bonding or by adsorption. These components can be associated with the particle dispersion that remains fixed at the application site after application to the absorbent support. For example, a suspension of cells having a specific receptor on the surface, such as Staphylococcus aureus cowan I (Staphylococ
cus aureus Cowan I) Latex particles carrying cells or binding partners with biological affinity bound to the surface are suitable for immobilization on paper matrices. The components of the test strip and the unbound components of the device bound to the pipetable support can also be dried on the absorbent matrix of the element by air drying. No lyophilization step is required. [0016] Some test examples are described as example embodiments that can be considered independent of the label used. For simplicity, only the detection of a single analyte by the diagnostic device is described. The following two embodiments, based on the principle of an antagonistic immunoassay, are used to determine whether the analyte has only one binding site with biological affinity or that several analytes with biological affinity have The case where only one binding site is used will be described. The solid phase binding partner binds covalently or by adsorption to a support material in the solid state action region. The analyte solution renders a predetermined amount of the label contained in the diagnostic agent mobile. The two components migrate into the working sector containing the solid phase binding partner and antagonize for binding with the solid phase binding partner. If the proportion of the analyte is higher than the labeled analyte, the labeled analyte will hardly be bound. If it is low, a large amount of labeled analyte will be bound. The solid phase binding partner is housed as an unbound component in the working region, in front of the solid state working region. The approaching solvent front carries it to the solid-phase working area where it becomes bound. The solid phase binding is created by a binding system having a biological affinity independent of the analyte binding system. The binding partner conjugated to biotin binds to avidin bound to the support. Immunoglobulins as binding partners, such as IgG, bind S.V. to a support via its Fc component. It may be immobilized on Aureus Protein A or bound by another species of solid phase antibody directed non-idiotypically to the immunoglobulin. As mentioned above, the analyte and the labeled analyte compete as components of the diagnostic agent for binding to the solid phase binding partner during the treatment time. Some of this antagonism occurs for dissolved solid phase binding partners, and some for solid phase binding partners already bound to the solid phase. If two binding points with different specificities are present in the analyte, several embodiments of the diagnostic agent based on the principle of a sandwich immunoassay are possible. Two examples will be described below. When the solid-phase binding partner is bound covalently or by adsorption to the support material in the solid-phase working region, the binary complex formed between the bound and the labeled binding partner migrates with the solvent into the solid-phase working region. It then reacts with the solid phase binding partner to form a ternary complex bound to the solid phase, which can be detected through the label of the first binding partner. Excess labeled binding partner is removed by the solvent into the next working area. If the solid phase binding partner is present in an unbound form in the diagnostic agent and becomes mobile by the solvent, the two reactive components of the analyte having biological affinity include the analyte Simultaneously or sequentially, the ternary complex reacts with both reaction components and the resulting ternary complex is then converted to a solid phase active region (where, as described above, it has a second system having a biological affinity independent of that of the analyte). (Which becomes bound to the solid phase via the solid phase). To illustrate the above-mentioned embodiments and other embodiments based on the immunometric test principle, the principle of indirect antibody detection or the ELA (enzyme-labeled antigen) principle of the immunoassay, the summary table I Illustrates, by way of example, the distribution of the components of the agent therein and the composition of the solid phase complex after completion of the reaction, the amount of which is a measure of the concentration of the analyte in the sample. [Table 1] A fully integrated test strip that works according to the principle of heterogeneous immunoassay with solid-phase detection is not only feasible in principle, but is also evaluated in less than one hour It was found that normal RIA and ELISA quantification and sensitivity were achieved. The detection of a trace component of about 10 ⁻¹² mol / liter becomes possible at room temperature in a reaction time of 30 minutes or less, and the required amount of a sample is, for example, 10 ⁻¹⁶ mol corresponding to about 1 pg. However, the above arrangement also allows for testing of lower sensitivity requirements. Standard curves for 20-30 were obtained when the evaluation was performed using a Sanoquell reflectometer (from Quelle). Chromatography time for test elements containing full coloration is 1
Does not exceed 6 minutes. The assessment can also be performed visually. When HCG was used as the analyte, the starting point of the measurement range in the example using glucose oxidase and peroxidase labels bound to the solid phase was 0.3 ng / ml (corresponding to 3 U / liter). In the following examples, the application of the principle of the antagonistic dual antibody test is shown as a specific example.
In this test configuration, the four components need to be reacted sequentially for the measurement reaction and separation steps, and the reaction times and concentrations of the reaction components are critical values. This example is not to be construed as limiting the invention in any way, but is merely used to further illustrate the invention. EXAMPLES Fully Integrated Enzymes for HCG Detection with an Integrated Chromogenic Substrate System
Immunochemical device. 1.1 Reagents 1.1.1 HCG-peroxidase conjugate HCG having a specific activity of about 3000 U / mg was obtained from Organon. Peroxidase from horseradish was purchased from Boehringer Mannheim (catalog number 413,470).
). Hetero - bifunctional reagent N-.gamma.-maleimidobutyryloxy succinimide of (GMBS) from Behring Diagnostics, Inc., and Tanimori et al 19 1983 ". J.Imm.Meth 62, 123~131" as described in With HCG. 2-Iminothiolane hydrochloride (Sigma, cat. No. I 6256) was reacted with peroxidase by the method described by King et al. In "Biochemistry" 17 , 1499-1506 in 1978. Conjugates were prepared from GMBS-HCG and iminothiolane-peroxidase by the method described by Tanimori et al. Ultrog crude joint
Purified by gel chromatography on el ACA 44 (LKB). The fraction bound with about 1-2 peroxidase molecules per HCG molecule was used for the test. The conjugate was diluted with Enzygnost IgE incubation medium from Behringwerke (order no. OSD) (hereinafter simply referred to as incubation medium). 1.1.2 Antibodies Antibodies to HCG were obtained by immunizing rabbits, and antibodies to rabbit-IgG were obtained by immunizing goats. The IgG fraction was isolated from serum by ammonium sulfate precipitation and anion exchange chromatography, and further purified by immunoadsorption. The method used is described in "Immunologische Arbeitsmethoden (Immunology Research Method)" (1984), edited by Helmut Friemel. Anti-HCG antibodies were finally diluted in the conjugate dilution buffer described above. 1.1.3 Glucose oxidase Glucose oxidase from Aspergillus niger was obtained as a solution containing 300 U / mg (Serva, catalog number 22,737). Glucose oxidase was finally diluted in the incubation medium. 1.1.4 Glucose and tetramethylbenzine α-D-glucose and tetramethylbenzine hydrochloride were obtained from Serva (catalog numbers 22,720 and 35,926, respectively). 1.2 Preparation of the Device The sheet-like working area was prepared as follows: The mobile phase application zone was prepared by cutting Kalle sponge fabric to a size of 20 × 6 mm. This is a synthetic sponge of regenerated cellulose compressed in the dry state. It was impregnated with 50 mg of glucose and 0.75 mg of tetramethylbenzidine hydrochloride per ml of water and dried in a stream of air. The conjugate, anti-HCG antibody and glucose oxidase (25 μl each)
1 / ml, 100 μl / ml and 5 μl each at a concentration of 0.1 mg / ml) were applied at equal distances to a piece of 45 × 5 mm MN No. 1 paper (Macherey & Nagel) in succession and air-dried. A sheet of Schleicher & Schuell 597 paper measuring 5 x 5 mm was coated in a conventional manner with anti-rabbit IgG-antibody as solid phase zone. This was done by binding the antibody to paper activated with cyanogen bromide (Clarke et al., “Meth. Enzymology” (19).
79), Vol. 68, 441-442). A 20 × 5 mm piece of Schleicher & Schuell 2668/8 paper was used as the absorption zone. The four pieces of paper were successively attached to a plastic ribbon with a double-sided adhesive tape ("Tesaband" manufactured by Beiersdorf) while overlapping by 0.5 to 1 mm to form a test strip having a width of 5 mm. 1.3 Performing the Test For each example, the test was performed by applying 200 μl of HCG diluent in the incubation medium to the fabric. 1.4 Results Chromatographic development and automatic color development of the test elements were completed after 15 minutes at room temperature, and the evaluation could be performed visually or by means of a reflectometer. The following values were obtained when the solid phase zone (# 597 paper) was evaluated using a Sanoquell blood glucose evaluation device manufactured by Quelle. [Table 2] Using a Behringwerke Rapimat urine test strip evaluation device, the following values were obtained for the same test strip. [Table 3] The test strip assembly shown here comprises glucose oxidase and anti-HC
This can be achieved even if the G antibodies are located in the same zone. Test strips so short give results after about 10 minutes.

Claims (1)

Claims 1. An analytical device for detecting or measuring one component (hereinafter, referred to as an analyte) of a pair of combinations having immunological binding characteristics existing in a liquid, comprising: The device is formed from a solid support and a sheet-like zone consisting of two or more strips, one or two arranged one behind the other and in mutually resorbable contact via their edges A chromatographic analysis device in the form of a sheet, comprising a mobile phase application zone (MPAZ) at one end of the device, an absorption zone (AZ) at the other end, and a further absorbent zone in the middle thereof. In the zone, a plurality of reaction components capable of forming an immunological interaction with the analyte are spatially separated from each other, and one reaction component is covalently bonded or adsorbed to the MPAZ.
And is immobilized in a solid phase zone (SPZ) located between and in contact with AZ, and the labeled reaction component is located unbound in the MPAZ or in the zone between MPAZ and SPZ. In one analytical device, the device contains all the reaction components and reagents in a dehydrated state, and the sample
Providing said analytical device wherein the MPAZ is such that the liquid controlled by the capillary force in the subsequent zone releases at least enough liquid to reach the end of the AZ; Applying a liquid sample containing the analyte, allowing the sample to reach the end of the AZ under control of capillary force, and measuring the amount of label in the SPZ, Immunological measurement method. 2. The reaction generated in the analysis device is based on a principle of an immunological detection reaction, an antagonistic immunoassay or a sandwich immunoassay, an indirect antibody detection with a labeled antibody, or an antibody detection with a labeled antigen. the method of. 3. A fluorophore wherein the labeling agent is detected or measured directly or after the addition of a reagent present in said analytical device, or detected directly or after the addition of a further reagent. 2. The method of claim 1, wherein the fluorophore to be measured is formed from a labeling agent by addition of a reagent present in the analytical device. 4. The method according to claim 1, wherein the labeling agent is a compound capable of generating chemiluminescence when excited, and the chemiluminescence can be detected or measured after addition of a reagent present in the analysis device. 5. The method according to claim 1, wherein the labeling agent is an enzyme whose activity can be measured using a reagent present in the analysis device.