FI90800C - Biospecific assay method and product for assay - Google Patents

Description

90800

BIOSPHERIC DETERMINATION METHOD 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 assay-specific components of the biospecific reaction in a ready-to-use form, wherein the other assay-specific components are dried to a ready-to-use form.

The invention also relates to a product for biospecific assays which encapsulates a reaction well containing the solid phase i component and other assay-specific components of the biospecific reaction in a ready-to-use form in a ready-to-use form for performing an assay.

BACKGROUND OF THE INVENTION

Especially easy-to-use, fast immunochemical 20 måMrity has 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 mono-25 clonal antibodies in particular have been applied in increasing numbers.

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become routine in the range of both competitive and non-competitive assays, and the traditional test tube has often been replaced by alternative solid phases.

2

In a routine hormone test, 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 more recently 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 only as a vessel or as a solid phase for the immobilized assay component. All other assay components must be added separately to the individual microtiter wells on 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 by the company. The number of components will, of course, vary according to the mSS company and its principle. As a result of the TSstM, the user of such a product has to perform a large number of operations and pipetting steps to perform the assay.

20 These factors alone make microtiter plate-based immunoassays cumbersome 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 in which freeze-drying or freezing is used to obtain 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,

II

3 90800 obtained by allowing a solution or suspension containing the second component to fall freely through a bath, a 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 eliminates many of the 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 a ready-to-use slit.

According to the method of the invention, the assay is performed with an additional sample or standard reaction well in which the other specific components of the biospecific reaction are dried to a ready-to-use form on a solid phase with a separate substrate placed in the reaction well, such as plastic, nitrocellulose or cellulose.

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 clean 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 according to 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 solid phase separate from the support well, such as plastic, nitrocellulose, cellulose or glass fiber.

4

According to an embodiment of the EraMn, the plate-shaped product may be an immunoassay product (putty) comprising 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 immunomaterial product contained a ready-to-use microtiter plate containing 96 individual assays.

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

The invention satisfies the following requirements: 1. All specific reagents are included in the ready-to-use form of the product (kit) = in a microtiter plate 2. Specific reagent containers are not included in the product 20 3. 5. Both competitive and non-competitive immunoassays such as back-titration assays can be used. 6. The product can also be used for nucleic acid hybrid disinfection assays.

II

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

5 DETAILED DESCRIPTION OF THE INVENTION

The method of the invention can be applied to both competing and non-competing specific immunoassays, including, for example, viral antibody assays and serological assays, or also to nucleic acid hybridization assays or receptor-10 assays.

All immunoassays are performed on a ready-to-use micro-titer plate, minimizing processing time and optimizing erty-type assay, automation, repeatability, and standardization.

The invention is described as follows:

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

Figure 2. shows a ready-to-use microtiter plate with 20 rows lf 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 of the reaction Ivor ivej a,

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 includes reaction wells as shown in Figures 1 and 2. As long as the reaction well row 6 is capable of retaining the immobilized component and, if necessary, assay-specific additional components, the shape or material of the microtiter plate as such does not limit the performance of the invention. The microtiter plate 5 containing 96 wells can either be cast in one piece or it can be assembled from 1 to 12 microtiter parts, one part 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 determination and its principle, which are known to a person skilled in the art who is familiar with the method.

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

In a non-competitive (non-competitive) assay, the addition of a labeled immunocomponent (antibody) to the microtiter wells of a coated reaction well row is such that the labeled component cannot bind non-specifically to the coated microtiter well. The labeled component is then 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 nMy 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. 35 Tate can be subjected to a single-step non-competitive immunoassay in microtiter wells of the li 90 90 reaction well row, which already contain all assay-specific components.

Ready-to-use assay-specific microtiter plates can be standardized already 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 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 kMytto-ready 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 country-specific microtiter plate can be applied to different types of mAssignments either roanally or in a tSys automated mAsary.

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 lines 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 at 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 of hTSH made by a ready-to-use micro-titer plate method using reaction wells containing a 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 well wall. In addition, the wells contain a filter paper disc dried with another anti-hTSH monoclonal antibody labeled with fluorescent europium chelate.

hTSH Assay The ready-to-use microtiter plate (product) contains the following steps: 1. Pipette 50 μΐ of assay buffer in large batches + 50 μΐ nSytett is added to each well of the 20 wells.

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

3. Wash the wells of the reaction well row (immunoassay completed).

25 4. A 200 μΐ solution is added to remove the fluorescent europium chelate from the well surface.

II

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 hTSH determination 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 a 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 the 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, causing a continuous line of tate leixna 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, round discs were punched from the well, centered on the 20 lines formed by the stamp. The diameter of the plastic disc was 3 mm and the volume of the 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 mSariation 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 under reduced pressure. Each strip was aspirated and washed six times. 200 μΐ of the measurement solution was added, and the strips were shaken slowly for three minutes to remove the fluorescent Eu chelate into the 10 solutions. Fluorescence was measured with a Platefluorometer, and the results are shown in Figure 4.

li

Claims (7)

A biospecific assay method such as a specific immunoassay, nucleic acid hybridization assay or receptor assay, specific reagent materials are used in a biospecific reaction with a solid phase component, the assay being carried out by adding a sample or standard containing both a reaction pit the ready-to-use solid phase component and the other components for the determination of the biospecific reaction in finished form, whereby the other components for the determination are dried in finished form for use, characterized in that the sample or standard is allowed in reaction the pit, in which the other components for determining the biospecific reaction are dried in finished form for use on a substrate placed separately from the solid phase in the reaction pit, such as on a plastic, nitrocellulose, cellulose or fiberglass substrate. 2. A 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). 3. A method according to claim 1 or 2, characterized in that the result is fed from the reaction row pit using fluorometry such as time difference fluorometry, photometry, luminometry or gamma or beta counter. A product intended for biospecific assays, such as specific immunoassay, nucleic acid hybridization assay or receptor assay, which comprises a reaction group containing a solid phase component and the other components intended for the assay to prepare the ready-to-use biospecific reaction reagent in the product for carrying out the determination, wherein the other components for the determination of 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 the determination of the biospecific reaction are dried in finished form. for use on a substrate placed separately from the solid phase in the reaction pit, such as on a plastic, nitrocellulose, cellulose or fiberglass substrate. The product of claim 4, characterized in that the reaction pit forms with other like pits at least one row of reaction pits (A-H; 1-12). 6. A product according to claim 5, characterized in that the product is in the form of a microtiter plate and consists of one or more connected pieces, the plate consisting, for example, of 8 rows of 12 pits or of 12 rows of 8 pits when it reaches 90 degrees from the original cover. Product according to claim 5 or 6, characterized in that the year is for specific immunoassay, which is a competing assay, a non-competing assay, a virus-antibody assay or a serological assay.