FI90800B - Biospecific assay method and product for assay - Google Patents

Description

90800

BIOSPHERIC METHOD OF DETERMINATION AND PRODUCT FOR DETERMINATION

The invention relates to biospecific assay methods using specific reagent materials in a biospecific reaction with a solid phase component, wherein the assay is performed by adding a sample or standard 5 to a reaction well containing both a ready-to-use solid phase component and other biospecific reaction-specific assay-specific components. has been dried to a ready-to-use form.

The invention also relates to a product for biospecific assays comprising adding a reaction well containing a solid phase component of the biospecific reaction and other assay-specific components in a ready-to-use form to the product for analysis, wherein the other assay-specific components of the biospecific reaction are dried in the reaction well.

BACKGROUND OF THE INVENTION

In particular, easy-to-use, rapid immunochemical assays have long been in need on the market.

Conventional radioimmunoassays were initially performed in solution using test tubes and complex separation methods. Since the introduction of solid phase technologies, various markers have replaced radioisotopes and, in particular, mono-25 clonal antibodies have been increasingly applied.

These improvements have differentiated conventional immunoassays (e.g., Alternative Immunoassays, Ed. W.P. Collins, Wiley, Chichester 1985). Solid phase assays have become routine in both competitive and non-competitive assays, and the traditional test tube has often been replaced by alternative solid phases.

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In a routine hormone assay, a microtiter plate was introduced as a solid phase in 1984 when Wallac Oy (Turku, Finland) introduced DELFIAR technology to the market. Other commercial companies (e.g., Amerlite, Amersham International U.K.) have 5 subsequently introduced similar solid phases in similar routine experiments, although they have been based on other labeling technologies. To date, all immunoassays based on the use of microtiter plates use the plate either as a vessel only or as a solid phase for the immobilized assay component. All other assay components must be added separately to the individual microtiter wells of the plate. This results in commercial products (kits) based on the microtiter plate format having to contain a number of separate containers (bottles) containing the components required for the assay. The number of components will, of course, vary with the specification and its principle. As a result, the user of such a product has to perform a large number of steps and pipetting steps to perform the assay.

20 These factors alone make microtiter plate-based immunoassays difficult to use and difficult to automate.

U.S. Patent 4,017,597 discloses a product in which the first component is on a solid support and the second component is on a solid support, but is rendered unreacted by rapid freeze-drying of a solution containing this component.

U.S. Patent 4,162,003 discloses a product that uses freeze-drying or freezing to render components in an unreacted form at various locations.

Finnish publication 84301 has a separate detached body containing a second component, which is on top of the first component in the solid phase. However, this is also a freeze-dried component,

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3,90800 obtained by allowing a solution or suspension containing the second component to fall freely through a cold, water-immiscible liquid having a lower density than water, after which the frozen buttons thus obtained can be freeze-dried.

The disadvantage of freeze-drying is that it requires special equipment in the manufacture of a product for biospecific assays.

INVENTION IN BRIEF

The present invention overcomes many weaknesses and imperfections compared to microtiter plate-based immunoassays using non-reaction reagents, with the exception of the immobilized component, and compared to the aforementioned known solutions in which the components are in ready-to-use form.

According to the method of the invention, the assay is performed by adding a sample or standard to a reaction well in which the other assay-specific components of the biospecific reaction are dried to a ready-to-use form on a solid phase separated from the solid well, such as plastic, nitrocellulose, cellulose or glass fiber.

The use of ready-to-use specific reagents is easy in the form of a microtiter plate according to the invention. The only additional components required to perform the assay in a ready-to-use form are a sample or standard and a sample buffer that also includes a wash solution.

The product of the invention is characterized in that in the reaction well, the other assay-specific components of the biospecific reaction are dried to a ready-to-use form on a support separate from the solid phase in a reaction well, such as plastic, nitrocellulose, cellulose or glass fiber.

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According to one embodiment, the plate-shaped product may be an immunoassay product (putty) containing a microtiter plate (8x12 or 12x8 wells) in which each well row is coated with an immobilized solid phase component (reaction well-5 vi). Respectively, each microtiter plate contains 8 x 12 or 12 x 8 reaction well rows. Thus, the immunoassay product contains 96 ready-to-use microtiter plates containing individual assays.

Other amounts of 10 reaction well rows can also be applied to the above product. The immunoassay product may contain other plate impactors, including varying amounts of reaction digestate. In addition, the immunoassay product may contain several specific assays. Standards are provided separately, if necessary, and assay buffer, including wash solution, is provided in large batches.

The invention meets the following requirements: 1. All assay specific reagents are included in the ready-to-use form of the product (kit) = microtiter plate 2. Assay specific reagent containers are not included in the product 20 3. Manufacturer can perform analyte specific standardization 4. Analyze specific assays for product (kit) = microtiter plate assay, can be performed either manually or fully automated 25 5. Both competitive and non-competitive immunoassays as well as assay methods requiring back-titration can be used. 6. The product can also be used for nucleic acid hybridisation assays.

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5 90800 7. The product can also be used for receptor assays 8. Product (putty) = microtiter plate is especially suitable for either manual or fully automated batch type assays

5 DETAILED DESCRIPTION OF THE INVENTION

The method of the invention can be applied to both competitive and non-competitive specific immunoassays, including e.g. viral antibody assays and serological assays, or also to nucleic acid hybridization assays or receptor assays.

All immunoassays are performed on a ready-to-use microtiter plate, minimizing processing time and optimizing batch-type assay, automation, reproducibility, and standardization.

The invention is described as follows:

Figure 1. shows a ready-to-use microtiter plate in which rows A, B, C, D, E, F, G and H are reaction well rows,

Figure 2. shows a ready-to-use microtiter plate in which rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 are the reaction i vor i ve,

Figure 3. shows a standard plot and repeatability curve of an hTSH assay performed in accordance with the invention, and Figure 4. shows a standard plot and repeatability curve of an hCG assay performed in accordance with the invention.

The assay-specific microtiter plate contains reaction wells as shown in Figures 1 and 2. As long as the reaction well row 6 can retain the immobilized component and, if necessary, additional assay-specific components, the shape or material of the microtiter plate as such does not limit the performance of the invention. The 96-well microtiter plate 5 can either be cast in one piece or assembled from 1 to 12 microtiter sections, with one section containing either 8 or 12 wells. Alternatively, it can be made from individual cast wells mounted in a suitable microtiter plate-shaped frame.

The ready-to-use assay-specific microtiter plate is machined by coating the microtiter wells of the reaction well row with the required solid phase component of the biospecific reaction. The method and the components to be selected depend on the specific assay and its principle, which are known to a person skilled in the art.

The microtiter wells in the reaction well row also contain other assay-specific additional components of the biospecific reaction. The addition of ready-to-use additional components, such as labeled components, must take place at the production stage so that the product is ready for use when a sample or standard and sample buffer, including washing solution, are added.

In a non-competitive assay, the addition of the labeled immunocomponent (antibody) to the microtiter wells of the coated reaction well row must be such that the labeled component cannot non-specifically bind to the coated microtiter well. In this case, the labeled component is quantitatively dried on filter paper, from which a disc containing a certain amount of label component is cut into each microtiter well of the coated reaction well row. The filter paper disc can be, for example, plastic, fiberglass, nitrocellulose or cellulose. In the assay, the sample buffer rapidly dissolves the dried label component on the paper and the paper disc as such is removed from the microtiter well of the reaction well row during the first washing step. Thus, a single-step non-competitive immunoassay can be performed in microtiter wells of the li 7 90800 reaction well row, which already contain all assay-specific components.

Ready-to-use assay-specific microtiter plates can be standardized at the manufacturing or production stage so that only controls and samples are analyzed when the product (putty) is used.

The applicable tracer in the ready-to-use microtiter plate-based product should be read directly from the reaction well. The invention is particularly directed to time-resolved fluoro-10metry and the use of lanthanide chelates as a tracer to provide either a qualitative or quantitative reading after an immunoassay.

As such, the invention does not preclude the use of other markers, such as radioisotopes, enzymes, conventional fluorophores, and chemiluminescence, if they are compatible with the ready-to-use reaction well microtiter plate concept described previously. It follows that the measurement is performed with either a beta or gamma counter, a photometer, a fluorometer or a luminometer.

20 The ready-to-use assay-specific microtiter plate can be applied to batch-type assays either manually or in a fully automated assay.

In connection with the manual method, for example, the following assay principle can be applied: 25 Non-competitive single incubation assay (product includes reaction well rows with microtiter well-specific filter paper disc)

Steps: 1. Pipetting the sample and buffer into the reaction well row 30 2. Incubation, shaking, at room temperature (or 35 ° C) 8 3. Washing the reaction well row (immunoassay completed) 4. Measuring the reaction well row

The following non-limiting application examples illustrate the operation of the invention: Example 1:

A non-competitive single-incubation time-resolved fluoroimmunoassay for hTSH performed by a ready-to-use microtiter plate method using reaction wells containing labeled antibody on a filter paper disc.

The wells of the reaction well row of the microtiter plate contain anti-hTSH monoclonal antibody immobilized and dry on the wall of the wells. In addition, the wells contain a filter paper disc dried with another anti-hTSH monoclonal antibody labeled with fluorescent europium chelate-15a.

The hTSH assay using a ready-to-use microtiter plate (product) includes the following steps: 1. Pipette 50 μΐ of assay buffer in large batches + 50 μΐ of sample into each well of the 20 wells.

2. Incubate the microtiter plate for 60 min. constantly shaking at room temperature.

3. The wells in the reaction well row are washed (immunoassay completed).

25 4. Add 200 μΐ of solution to remove the fluorescent europium chelate from the surface of the wells.

n 9 90800 5. Europium fluorescence is measured from each well of the reaction well row using time-resolved fluorometry.

Score:

Figure 3. Standard curve and repeatability curve 5 of the hTSH assay performed according to the invention.

Example 2:

A non-competitive single incubation time-resolved fluorescence assay for hCG performed in ready-to-use reaction wells containing labeled antibody on a plastic disk.

The anti-hCG monoclonal antibody labeled with fluorescent europium chelate was diluted in a drying solution and the solution was applied to a plastic surface by feeding a constant flow through a stainless steel capillary tube. The capillary tube was in contact with the surface of the plastic film during application, thus causing a continuous line of label solution with a constant longitudinal label concentration. The fed solution was dried on the surface of the film with a strong air flow. After drying, the film was stamped into round discs centered with respect to the 20 lines formed by the stamp. The diameter of the plastic disc was 3 mm and the amount of dried solution on the disc was 0.2 μΐ. The discs were transferred to strip wells previously coated with anti-hCG monoclonal antibody and dried.

25 μΐ hCG standards were pipetted into the wells of the strip. 100 μΐ assay buffer was added to each of the 25 wells. The strips were incubated at room temperature for one hour with slow shaking. After the incubation step, the discs were removed from the reaction wells with a suction nozzle connected to a vacuum. Each strip was aspirated and washed six times. 200 μΐ of measurement solution was added and the strips were shaken slowly for three minutes to remove the fluorescent Eu chelate n

Claims (7)

A biospecific assay method, such as a specific immunoassay, nucleic acid hybridization assay or receptor assay, using specific reagent material in a biospecific reaction with a solid phase component, the assay being carried out by adding a sample or standard in a reaction pit containing the ready-to-use solid phase component and the other components for determining the biospecific reaction in ready-to-use form, wherein the other components for determination are dried in finished form for use, characterized in that the sample or standard is added in the reaction pit, where the other components for determining the biospecific reaction are dried in finished form for use on a substrate placed separately in the reaction pit, such as on a substrate of plastic, nitrocellulose, cellulose or fiberglass. Method according to claim 1, characterized in that there are several reaction pits in one row or more rows of reaction pits (A-H; 1-12). Method according to claim 1 or 2, characterized in that the result is measured from the reaction row pit using fluorometry, such as time difference fluorometry, photometry, luminometry or gamma or beta counters. A product intended for biospecific assays, such as specific immunoassay, nucleic acid hybridization assay or receptor assay, comprising a reaction group containing a solid phase component and the other components intended for the assay to complete the biospecific reaction for use in preparation for use. in the product for carrying out the assay, wherein the other components for determining the biospecific reaction are in the reaction pit dried in finished form for use, characterized in that in the reaction pit the other components for determining the biospecific reaction are dried in finished form. for use on a solid phase separately, in the reaction pit, placed in the reaction pit, such as on a plastic, nitrocellulose, cellulose or fiberglass substrate. 5. Product according to claim 4, characterized in that the reaction pit forms with other similar pits at least one row of reaction pits (A-H; 1-12). Product according to claim 5, characterized in that the product is in the form of a microtiter plate and consists of one piece or several assembled pieces, the plate consisting, for example, of 8 rows of 12 pits or of 12 rows of 8 pits, when turns 90 degrees from the original position. Product according to claim 5 or 6, characterized in that it is intended for specific immunoassay, which is a competing assay, a non-competing assay, a virus-antibody assay or a serological assay.