SE446666B - PROCEDURE AND DEVICE FOR DETERMINING A MOVABLE COMPONENT IN ONE IN A CONTAINER INFORTED LIQUID SAMPLE, WHICH THIS COMPONENT REACTS WITH IMMOBILIZED COMPONENT APPLIED ON A MATRIBLE WITH SIMILAR PROVISIONS - Google Patents

Description

446,666 chemical methods are indistinguishable from other substances which are usually present in the same reaction mixture. This means that only the disappearance of a reactive component from the solution or accumulation on the solid phase cannot be measured directly. Additional steps must therefore be taken to achieve a measurable change related to the degree of binding.

Two different main methods have been used so far. According to one, called competitive or indirect immunoassay, the immobilized component is present in a regulated amount and the moving component is present in an unknown amount. To the unknown amount of moving component is added a known amount of the same component, which has been labeled by the addition of a measurable substituent which does not interfere with the immunological reactive properties of the component. The labeling substance may be a radioisotope, a chromophore, a fluorophore or an enzyme. The amount of labeled component that binds immunochemically to the solid phase depends on the amount of unlabeled component in solution competing for the same binding sites. The more unlabeled component that is present, the less amount labeled. According to the second main method, called the sandwich method or the direct method, the solid phase containing a certain amount of immunochemically bound mobile component is subjected to the action of a reagent which can also be immunochemically bound to the solid phase but only in places already occupied by the immunochemically bound _moving component. The reagent may be labeled, e.g. with a radioisstop, a fluorophore, a chromophore or an enzyme. The amount of bound labeled reagent is a direct measure of the amount of bound mobile component, which in turn is a measure of the amount of mobile component initially present in the reaction mixture.

If the label is a radioisotope, the method of radioimmunoassay is called whether competing or non-competitive techniques were used. If the label is an enzyme, the method is called enzyme-linked immunoassay. The amount of enzyme-labeled reactant is measured according to any suitable method for measuring the activity of the enzyme used.

The following are examples of other solid phase reactions of the type described above. Immunoradiometric analysis for quantitative determination of an antigen is performed by first reacting a known excess of labeled antibody with the unknown amount of antigen in a homogeneous phase reaction. Excess immobilized antigen is then added to bind the unreacted, soluble, labeled antibody. The amount of unknown antigen is determined by measuring the difference between the total amount of labeled antibody and the amount that binds to the solid phase. This method gives direct quantitative results only with a monovalent antigen, ie. an antigen that can bind only one antibody molecule.

Enzyme-catalyzed reactions are conveniently carried out in solid phase. An enzyme immobilized on a solid phase matrix can be used for quantitative determination or qualitative detection of the substrate for the enzyme in a sample of biological material. Lactic acid in serum can e.g. determined using a matrix coated with lactic acid dehydrogenase. Similarly, urea can be determined using a solid phase matrix carrying immobilized urease. In addition to clinical applications, enzyme analyzes can be used for quality control in industrial processes and also for carrying out process steps. Immobilized penicillinase can e.g. be used to monitor the quality of a penicillin during its manufacture.

Immobilized proteases or nucleases can be used to remove or inactivate contaminating proteins or nucleic acids.

The insertion matrices of the invention can be conveniently removed at any suitable reaction step so that the degree of the desired reaction can be easily controlled.

The presence of a clinically important enzyme in a sample of biological material can also be detected by using a substrate for the enzyme immobilized on a solid phase matrix. An example of such an assay is the method described in U.S. Patent Application 795,497. Another example of the use of immobilized substrates in enzyme assays is the determination of lysozyme, in which radiolabeled Micrococcus lysodeikticus is covalently bound to the surface of a solid phase matrix.

Other examples of useful solid phase reactions are specific binding reactions with certain proteins. Examples which may be mentioned β-dactoglobulin which specifically binds folic acid, specific receptor proteins capable of binding hormones such as the receptor substance isolated from breast cancer tumor cells in rats which specifically bind prolactin, and the variety of plant proteins, e.g. concanavalin A, which can specifically bind certain carbohydrates.

Conventional chemical reactants can be used in solid phase reactions. One can e.g. use reactants capable of forming colored complexes, such as the formation of glycosyl derivatives or diazo coupling to a reagent immobilized on the surface of a solid phase matrix, either separately or in combination with an enzyme-catalyzed reaction, so that a color change takes place on the surface of the matrix. Even ion exchange reactions can be conveniently carried out using a solid phase matrix according to. the invention. The above examples are illustrative only, and other possibilities are obvious to those skilled in the art.

In such solid state techniques, the reagent (s) used are usually immobilized by coating on or bonding to the solid phase material, either by adsorption or by covalent bonding, after which the solid phase material is immersed in the sample to be analyzed. Various methods for coupling reagents to solid phase materials are known; see e.g. U.S. Patent Nos. 3652,761, 3,879,262 and 3,896,217.

Non-limiting examples of commonly used solid phase materials include glass tubes and polymer tubes which are coated with one or more reagents on the inner surface; micro- and macro-beads made of polymers, glass or porous materials.

Immunochemical analyzes are very useful in medical research and in making diagnoses. These assays are very specific because antigen-antibody responses are highly selective. The antigen-antibody binding is very solid, so that once the binding reaction has taken place, the limit of detectability is determined by the ability to detect the label substance. Immunochemical analyzes are extremely versatile because they can be used to determine specific substances selectively against a background of chemically similar substances. Due to these beneficial properties, extensive work has been done to improve the practical implementation, sensitivity, accuracy, speed and usefulness of immunochemical analyzes. The development of solid phase immunoassays has been one of the most important advances in this field.

One of the advantages of solid phase systems is that the reaction product or products can be separated from the reaction solution with relative ease, ie. by physical removal of the solid phase material. In the case of non-solid phase reactions or homogeneous reactions, on the other hand, a homogeneous solution is usually obtained which requires more complicated separation methods.

The introduction of solid state technology has made it possible to implement new processes, which have been extremely difficult to implement in a free solution. An example of this is the sandwich technique described above.

This technique would require a large excess of one of the reactants to be able to be carried out in homogeneous solution. More importantly, separation of the first antigen-antibody complex from a homogeneous solution requires the use of complex physicochemical methods, especially if the antigen is relatively small compared to the antibody and the molecular weight difference between the free antibody and the complexed antibody is small. When using solid phase systems, on the other hand, the separation process is extremely simple. One of the main advantages of the solid state technique, namely the ease of separating the solid and liquid phases, is maximized in carrying out the process according to the invention by using extremely simple means for separating the phases. This is described later in the description. Although solid state technology in theory has many advantages over free solution analysis or homogeneous systems, it does have some limitations, mainly due to the solid state configurations used heretofore. Since at least one of the reagents in a solid phase system is effectively immobilized by binding to the surface of the solid phase, the reaction rate in solid phase systems is ::: 1íí: ïi :: šr: nä: a: i: 0ï0E fi n: d systems (free solutions). Furthermore, there is the surface of the phase whereby the: nananb 'âeñgens which can be blended on the solid' maxlmlma fl åd b @ r0P on the specific surface, On Peaåensets purity 00h Then the Specific procedure used for ä:: lïïšße fi on the surface. optimally, as much of the surface of the beer as possible should be coated so that the reaction rate increases and the reaction time decreases. The first used solid phase systems were test tubes coated on Ü :::::::: ¿e§á ::: ne :: ï :: lKïa commercial coated tubes can be mentioned USA; "Rian" from New En ~: ned šstruments, Sunnyvale, California, seen, USA. and they concern sos: n K uclear, Northßillerica, Massachuten 3 867 gl? Although beïa es are torn in the U.S. patent. gda tubes have proven to be useful, they do not provide all the benefits that can be achieved with solid phase systems. A major disadvantage is that the surface / volume ratio is relatively small, and the reaction kinetics can be further hindered by the reactive surface being coated at the boundary of the volume of the solution, which limit can be relatively far from the main mass of the solution. The average distance between the moving reactants and the reactive surface is therefore large. In addition, each test tube must be coated; sepa_ 446 666 6 under static conditions, and this condition means that the amount of coating material varies from pipe to pipe, which results in varying analysis results. The applied coating may be non-uniform and also discontinuous, so that some areas of potentially reactive surface may lack coating while others may be too heavily coated to achieve optimal reactivity. In both cases, the surface area available for reaction with the moving component is reduced, which results in reduced sensitivity and reproducibility. The batch method for coating pipes is also relatively expensive. Reactions performed in coated tubes are subject to defects caused by convection in reaction fluidity. Convection can cause the results to vary by as much as ten power.

Attempts to improve coated pipes have led to a number of systems with an increased surface / volume ratio of the solid phase system. Thus, very pleated surfaces have been created, the required liquid volume has been reduced and surfaces of finely divided material have been produced.

The "SPAC" system from Mallinkrodt Chemical Company is a coated pipe system with pleated surfaces that provides increased specific surface area. The pipes are also provided with a detachable lower section, which can be batch coated in order to achieve greater uniformity from pipe to pipe. As a result of this batch coating of the pipe bottoms, both the outside and the inside of the pipes will be coated. This makes it difficult for the laboratory technician to work with the tubes without coming into contact with the coating material on the outside, and valuable immunological reaction components are lost. The pleated surface is said to increase the available reactive surface 3-4 times. However, the reactive surface remains at the periphery of the solution, and optimal geometry is not obtained with respect to the average diffusion distance from the solution to the reactive surface. Due to the special structure of the surface, it can be difficult to wash the surface free of contaminants. The "SPAC" system, like the pipe system, is generally sensitive to convection currents which can cause large errors (see above).

The convention can be reduced by carrying out the reaction in a constant temperature bath. However, this technology requires additional equipment. In addition, when determining hapten antigens, the system is not optimal if the reaction is carried out at 57 ° C according to the manufacturer's recommendations. It has been shown that an increased temperature in certain antibody-hapten reactions tends to increase the dissociation rate of the antibody-hapten complex relative to the rate of formation; see Smith, T.W. and Skubitz, K.M. , Biochemistry, 14, 1496 (1975) and Keave, P.M., Walker, W.H.C.

Gauldie, J. and Abraham, G.E., Clin. Chem. 22, 70 (1976).

Various types of solid phase matrices intended to be introduced into the reaction fluid have been described. U.S. Pat. No. 3,951,748 discloses e.g. a pleated or sponge-like matrix intended to be introduced into the sample solution. This matrix has a relatively large specific surface area, but it can be an answer to wash properly after the reaction is completed. In addition, such a system is in practice limited to the use of such reagents and reactants which are relatively easy to elute from the sponge-like matrix. Furthermore, fungal-like matrices have a tendency to react completely with only a part of the reaction fluid, i.e. with the part penetrating into the pores.

Another type of matrix, in which use the reaction fluid is spread in a thin layer over the coated matrix surface, is described in U.S. Pat. Nos. 3,826,619 and 3,464,798.

Both of these publications describe a combination of a container and an insertion matrix in such a form that it squeezes out the reaction liquid in a thin layer between the container wall and the matrix surface. The insertion matrix must fit exactly into the container, and the volume of reaction liquid must be carefully controlled, as variations would adversely affect the reproducibility of the assay. The device according to USP 3,826,619 is intended only for qualitative, direct immunological analysis. Since the reaction solution is squeezed into a thin layer of the insertion matrix, the reaction volume must necessarily be small, and USP 3,826,619 states that the device described is intended for small volumes of undiluted serum. One of the disadvantages of this type of assay is that the rate of antigen-antibody responses may vary due to variations in the pH of the undiluted serum; this pH value can vary between 6 and 9 in clinical trials. The pH can be adjusted by adding buffer, but buffer salt concentrations higher than 0.1M tend to dissociate antigen-antibody complexes. Therefore, a large volume of low ionic strength buffer must be used to accurately control the pH, and this may increase the reaction volume to an unacceptable degree. Errors due to inappropriate pH value can be tolerated in qualitative analyzes, in particular in the analysis of samples containing the substance to be detected in high concentration, but not in such quantitative analyzes as the present invention relates to. If dilution with buffer is required, a low concentration of the substance to be detected may lead to dilution beyond the detection level, which may lead to false negative results. An embodiment of the insertion matrix according to USP 3,826,619 is a fin-provided insertion matrix, which is somewhat similar to the 4-fin embodiment of the present invention. This known matrix is intended for qualitative analysis, when large amounts of serum are available, but it is not stated that it can be used in any other way than the round or conical embodiment, i.e. for thin layer analysis. The devices described in USP 3,826,619 have not been manufactured commercially.

Another known matrix type is "StiQ" from International Diagnostic Technology Corporation, Santa Clara, California, USA, which is intended for use in the assay method described in U.S. Patent 4,020,151. A disc-shaped, uncoated insertion matrix is used. of a material that can adsorb proteins from serum. The limitations of this system are not only due to unfavorable geometric conditions or unfavorable surface / volume ratios but are mainly due to problems associated with the initial adsorption step, such as the presence of interfering substances and difficulty in obtaining measurable adsorption of components present. in low concentration.

Another attempt to increase the area / volume ratio by decreasing the reaction volume is described by Friedel, R, and Dwenger, A. in Clin. Chem. 21, 967 (1975). In this case, capillary tubes are coated on the inside with a specific adsorbent, and the reaction mixture is introduced into the capillary tubes.

One system that provides a large specific surface area per unit volume are coated microglass beads, such as "Immo Phase" from Corning Glass Works. This system is an example of the use of finely divided particles. A large coated surface is used and a high reaction rate is achieved. Due to sedimentation of the particles during the reaction, an optimization of this system requires that the test tubes, which during the reaction contain the particles, be closed and shaken vertically during the reaction in order for all surfaces to come into contact with the reactants. In addition, the use of particles requires several centrifugations and washes in order to obtain a complete separation of the immobilized product from the reactants in the solution. Surfaces of glass particles have the further disadvantage that there is a greater non-specific binding of proteins: iii glass than: 111 plastic.

Previous attempts to improve solid phase matrices, such as coated tubes, have usually led to some improvement in reaction rate or reaction time, but such improvement has usually been accompanied by a more cumbersome assay procedure or reduced flexibility. According to the present invention, both a larger surface area / volume ratio and an improved reaction kinetics are achieved. In addition, the versatility is improved and the analysis procedure is simplified.

As an example of the prior art, mention may also be made of U.S. Pat. No. 3,206,602, which, however, does not describe a solid phase matrix.

According to the present invention, there is provided a method of determining a movable component in a liquid sample inserted into a container, wherein the movable component reacts with an immobilized component on a handle-shaped insert immersed inside the liquid sample. This method is characterized in that the immobilized component used has been applied in the form of a fixed uniform coating of at least 8, preferably 8-18 from the handle of the insert radially projecting fins with smooth surfaces and with large dimensions in relation to the interior of the container space, that the average diffusion distance of the molecules of the moving component to the phenyls coated with the immobilized component becomes considerably smaller than the average diffusion distance to the inner surfaces of the container in the absence of the insert; and measuring a chemical, enzymatic or immunochemical change produced by the reaction of the components on these phenytes as a measure of the concentration of the moving component in the sample.

The invention also relates to a device for determining a moving component in a liquid sample, comprising a container for the liquid sample, preferably a test tube, and a handle-provided insert immersable in the liquid sample in the container, which carries an immobilized component, intended to react with the moving component. - ten. This device is characterized in that the insert for determining the concentration of the movable component comprises at least 8, preferably 8-18 fins projecting in different directions from the handle of the insert, which have smooth surfaces, support the immobilized component in the form of a fixed uniform coating and have such large dimensions in relation to the interior space of the container that the average diffusion distance of the molecules of the movable component to the phenyls coated with the immobilized component becomes considerably smaller than the average diffusion distance to the inner surfaces of the container in the absence of the insert.

The advantages of the invention are that time errors in the initiation and interruption of the reaction are eliminated, further that the reaction rate is high, that errors in volume transfer are reduced, that the measurement errors become smaller at a given sensitivity, and that the manipulation is simplified. The high reaction rate that can be achieved with the preferred embodiments makes it possible to perform immunochemical analyzes at room temperature or lower temperature, which can be advantageous compared to higher temperatures for equilibrium reactions. The device according to the invention is simple to manufacture and consequently inexpensive.

Many different matrices can be constructed according to the principles set forth in the present specification. The essential features are that the matrix comprises a handle and a plurality of substantially smooth, flat or curved surfaces attached to the handle, these surfaces being arranged in such a way and having such a shape and size in relation to the reaction liquid, that introduction of the matrix therein, the average diffusion distance of the molecules of the moving component to the matrix surfaces decreases compared to the average diffusion distance to the inner surfaces of the reaction vessel in the absence of matrix. The specifically described embodiments are given by way of example only and are not intended to limit the invention. A given matrix shape may but need not be designed in accordance with the size and shape of the reaction vessel into which the matrix is to be inserted. Furthermore, no fitting is required inside the reaction vessel.

The matrix must, of course, extend substantially along the entire height of the liquid sample. In some systems, the matrix surface preferably extends above the surface of the reaction liquid, thereby achieving a substantially constant geometric relationship along the entire height of the liquid sample and also ensuring that the same geometric ratio will prevail regardless of any changes in liquid volume.

Preferred devices of a given size are e.g. equally suitable for different analyzes where the reaction volumes differ by up to 3 times.

An alternative embodiment of a solid phase matrix according to the invention useful in chemical, enzymatic and immunochemical analyzes is particularly useful in connection with automatic pipetting equipment. This matrix can also be used to perform standard manual batch analyzes. The matrix has a closed support surface which delimits a well-like volume, in which a liquid sample can be applied when the matrix is placed in a container, such as a test tube. A multi-ital oriented member preferably forms a unitary piece together with the closed carrier surface. The innermost end of these inwardly directed members defines a centrally located cylindrical volume, into which an automatic pipetting device for inserting and aspirating liquid samples can be inserted.

Use of the preferred fin-provided matrix has in some cases resulted in an unexpected and as yet unexplained increase in the reaction rate beyond what could be predicted on the basis of the specific surface area. Following the discovery that geometric factors affect the reaction rate, it has become important to ensure uniform solid-phase geometry from one sample to another to ensure reliable results. The devices according to the invention provide such uniformity and also help to eliminate human and mechanical errors, such as variations in stirring speed, time errors, convection and the like.

In the process of the invention, a solid phase reaction is carried out using one or more reactants fixed on the surface of a matrix immersed in the reaction liquid. Examples are reactions carried out in test tubes in which matrices are inserted. The reaction is started by inserting a matrix into a test tube containing the mobile component, and the reaction is stopped by removing the matrix. Optionally, the reaction can be restarted by re-introducing the matrix, or a second reaction can be started by introducing a new matrix carrying a second fixed component. The matrix may optionally be placed in a second test tube containing a reagent, or it may be directly transferred to a radioactivity counting chamber or to some other measuring equipment depending on the nature of the assay method.

The kinetics of solid phase reactions are more complex than the kinetics of homogeneous phase reactions. A detailed theoretical basis for optimizing the construction of the deposit matrix is not available. However, some basic facts of a general nature can be taken into account. The total area in contact with the solution is an important factor. The larger this area, the greater the amount of fixed component can participate in the reaction. An increase in the effective concentration of either component usually increases the overall reaction rate.

Since the amount of fixed component is determined, among other things, by the area of the solid phase, the reaction rate varies directly with the ratio surface / volume. According to the invention, an increased surface / volume ratio is achieved by means of an insertion matrix, which has a more acidic surface than the inner surface of the test tube. Another factor that possibly affects the reaction rate is the average diffusion distance between the moving and the fixed reactant. "Average diffusion distance" means the sum of the distances that each molecular moving component must diffuse in order to reach a fixed component along the shortest possible path, divided by the total number of such molecules. The matrices of the invention are designed so that the average diffusion distance between the molecules of the moving component and a component fixed on the surfaces of the matrix is considerably smaller than the average diffusion distance when the fixed component instead adheres to the inner surfaces of the reaction vessel. and no deposit matrix is present. The transfer of moving components to the reactive surfaces is assumed to be facilitated by a reduction of the average distance between the moving components and the reactive surfaces. Increased reaction rates have been observed when using the insertion matrices according to the invention (see exemplary embodiments).

In a number of cases, it has surprisingly been found that the resulting increase in the reaction rate was greater than what could be expected on the basis of the increased area / volume ratio. The reactions have also been shown to reach equilibrium faster when using finely equipped matrices than when using coated pipes. Although these phenomena are not well characterized or qualified, they appear to be observed more often and more clearly when using finely equipped matrices with a larger number of fins.

The increased reaction rate and the rapid attainment of equilibrium are observed with sufficient frequency (although these phenomena do not always occur) to justify the assumption that geometric factors other than the specific surface of the matrix can significantly affect the rate of solid phase reactions. The use of matrices according to the invention is therefore expected to give favorable reaction rates for solid phase reactions in general. It is readily appreciated that practical considerations may determine an upper limit on the number of separate elements, such as fins, which may be attached to the matrix handle. The elements should e.g. do not sit too tightly, because then capillary forces can cause the reaction mixture to be retained between the elements, which makes washing and drainage more difficult. If a fin-provided rod is to be produced by molding, the number of fins can also be limited by the possibility of manufacturing a mold with the desired number of fins.

The upper limit imposed by such considerations depends on the size of the matrix and the purpose for which it is to be used. For matrices intended for use in reaction volumes of the order of 1 ml, the preferred number of fins is at least 8. In a particularly preferred embodiment, 18 fins arranged radially around a central handle are used.

The invention also brings other advantages. The increase in the total area makes it possible to carry out quantitative determinations within a wide concentration range for the moving component due to increased detectability in the lower part of the concentration range and increased binding capacity, so that a proportional response is possible at higher concentrations of the moving component. In addition, the matrices according to the invention can be used for carrying out quantitative analyzes according to the direct method (sandwich method). For this use, the increased binding capacity of the matrices of the invention is necessary for immobilizing materials over the full range of possible concentrations, more than is required for simple "yes-no" attempts (see U.S. Pat. No. 3,826,619). According to a preferred embodiment of the invention (see Example 7), antigen concentrations can be quantified within a wide range by immunoassay according to the sandwich method. In competing-type immunoassays, the range of analysis is determined by a complex interaction between the matrix geometry, the amount and distribution of antibody immobilized on the matrix surface and the immobilization method. The number, location, size and shape of the surface elements of the matrix affect the kinetics of the immunochemical reaction, which in turn affects the amount of immobilized component required to provide a differential response to the amount of moving component to be measured. The stability of the bonded component and the uniformity of the distribution of this component are other parameters that affect the design of the coated matrix. Advantageous results are obtained in competitive immunoassay using coated matrices according to the invention in that it is possible to regulate all essential factors that affect sensitivity, assay area and reproducibility. In addition, the process of the invention uses a sufficiently large reaction volume for a serum sample to be diluted with buffer to control the pH and reduce errors due to variations in the pH or other factors, such as the protein concentration, from sample to sample. In addition, a constant sample volume can be achieved. The devices according to the invention are not limited to use in a single determined reaction volume, since they do not depend on fitting with the reaction vessel. The reaction vessel need not have a special shape or size. The matrix of the invention need not fit exactly into the reaction vessel or even touch the walls of the vessel. The matrix elements with smooth, flat or curved surfaces should be evenly arranged along the height of the reaction liquid and in some systems preferably extend above the liquid surface to geometric relationship between the molecules of the moving component and the matrix surfaces at different large volumes of liquid.

In both direct and indirect immunoassay, the required reaction time depends on the rate at which the reaction approaches equilibrium. In the process according to the invention, equilibrium is reached faster than in conventional analysis in coated test tubes, so that the required reaction time is shorter. In addition, when using a matrix according to the invention, one can very precisely start and stop the reaction. This advantage becomes significant when it is necessary to perform a large number of analyzes simultaneously. In such cases, the manipulation time required to start and stop the reaction is reduced to a minimum, and the reaction time can be regulated exactly for all the samples in a series. This is a great advantage over the use of coated tubes which must be decanted, sponge-like matrices which must be extruded to remove the last traces of reaction mixture, and particulate matter which must be centrifuged and decanted.

An additional advantage due to the increased reaction rate and the increased capacity of the matrices is that the same matrix can be used for both sandwich analyzes and competitive analyzes. When used for quantitative competitive analysis, the matrix of the invention provides a proportional reaction response within a wide concentration range.

The geometric configuration of the matrices according to the invention entails further advantages. As stated above, the use of the matrices results in a clear increase in the reaction rate which cannot be attributed to the increased surface area. Although this phenomenon has not been explained so far, it is believed that a series of flat or curved surfaces extending throughout the height of the liquid sample reduces the average distance between moving components and fixed components distributed on said surfaces and that this decrease results in a higher overall reaction rate. In addition to a higher reaction rate and associated advantages, the device according to the invention gives more uniform results. The geometric relationship between the movable and the fixed component is constant and reproducible from experiment to experiment in contrast to when using particulate solid phases, where the particles have a tendency to settle at a rate depending on the particle size and particle size distribution. The method according to the invention is less sensitive to convection defects, which is a major problem when using coated pipes. The devices according to the invention are provided with substantially smooth surfaces, which enable rapid drainage of the reaction mixture when the matrix is taken up from the solution. This property makes it possible to quickly interrupt the reactions, simplifies or eliminates washing steps and reduces any pollution risks when radioisotopes are used. This eliminates many possible human sources of error. It is preferred to use matrices with maximum surface evenness produced by molding, whereby the mold surfaces are polished to a mirror gloss. Maximum surface smoothness can be achieved by the matrix being formed of plastic in a mold with the surfaces polished to a mirror gloss. That advantages 446 666 16 are achieved with maximally smooth surfaces may seem unexpected, since uneven surfaces give a larger specific surface area. However, such maximally smooth surfaces are used in a preferred embodiment of the device to maximize the removal of reaction liquid from the matrix surfaces and minimize non-specific background interference.

From the above it appears that the design of a properly functioning matrix requires attention to all the factors and variables that affect the reaction to be carried out. In addition to providing a matrix that provides increased reaction rate, the operational advantage of minimal background disturbances and all other advantages of the invention, it is important to provide a coated surface with immobilized reactant distributed in such a way that its reactivity is maximum. The immobilized component should be distributed as evenly as possible over the surface. Gaps in the coating must be avoided; such gaps can be caused by e.g. air bubbles located on the matrix surface during the coating step. The molecules of the immobilized component must be exposed on the matrix surface and must not be buried in excess of the reactant or other carrier material. The immobilized component should preferably be strongly bonded to the matrix so that a significant amount of this component is not desorbed or otherwise removed during the incubation and washing steps.

Embodiments provided with a plurality of individual protruding surfaces, such as fins or pins, can be used to perform several analyzes simultaneously, if different fins are coated with different materials (different immobilized components). The embodiment shown in Fig. 3 is particularly suitable for such purposes.

The sticks or rods can be individually attachable and detachable so that a combination of desired analyzes can be performed in a single reaction step and then the results measured individually.

In addition to the above-mentioned advantages of the new deposit matrices in quantitative analyzes, there are also production advantages of commercial significance. In particular, the fixed component can be immobilized on a large number of matrices simultaneously, either batchwise or continuously. This provides uniform products at a low price.

An embodiment of the matrix according to the invention is suitable for use in combination with an automatic pipetting apparatus. 446 666 17 This embodiment lacks handles and instead has a support surface delimiting a cylindrical, well-like volume, which is filled with liquid sample when the matrix is placed in a test tube. A plurality of radially inwardly directed members have their outer end attached to the inside of the support surface.

The inwardly directed members and the support surface are preferably made in one piece by compression molding or extrusion. Alternatively, they may be made in separate pieces, which are attached to each other by any suitable joining technique, such as tongue and groove, or by means of adhesive. The inner ends of the members define a centrally located non-cylindrical volume, which makes it possible to quickly insert (and even remove) the probe of an automatic pipetting device all the way to the bottom of a test tube containing the matrix. The radially inwardly directed members are arranged in planes which intersect the central axis of the centrally located cylindrical volume. The larger the number of such members, the larger the reactive area and the faster the reaction takes place with the practical limitations discussed above in connection with the matrix in the form of a fin-equipped rod. Alternatively, one end of the matrix may be closed to form a tube with aligned means, the free ends of which define a central volume into which a pipette can be inserted.

The matrices according to the invention can also be used in industrial laboratories for carrying out chemical or enzyme-catalyzed routine analyzes (eg quality control) which can be carried out in solid phase systems. In addition, the matrices can be used in continuous or discontinuous processes, where it is desired to remove a specific substance from a reaction mixture. Matrices coated with ion exchangers can e.g. can be used to remove specific ions from solutions, and specifically adsorbent proteins immobilized on matrices of the invention can be used to remove impurities, such as trace metals, from production streams. Conventional catalysts can be applied to the surfaces of the described insert matrices for continuous conversion of reactants, treatment of effluents and the like. Matrices according to the invention intended to fit reaction vessels of suitable size can be used for processes on an industrial scale or laboratory scale. Solid phase reactions with gaseous reactants, vapors, aerosols, particle suspensions and the like can also be carried out. The matrices according to the invention can be particularly useful in flowing systems, in which it is desired to carry out reactions without reducing the flow rate or introducing a back pressure.

The invention is described in more detail below with reference to the accompanying drawing, in which Fig. 1 shows the preferred embodiment of a solid phase matrix according to the invention; Fig. 2 shows a modification of the embodiment of Fig. 1 comprising a plurality of holes for increasing the reactive area; Fig. 3 shows another embodiment comprising a plurality of downwardly directed pins; Fig. 4 shows an embodiment with concentric cylindrical surfaces; Fig. 5 shows an embodiment with a continuously projecting surface in the form of a spiral ramp; Fig. 6 shows an embodiment with discs arranged one above the other; Figs. 14a and 14b are photomicrographs of matrix surfaces taken in scanning electron microscopes.

Fig. 1 shows the preferred geometric shape of an insertion matrix 10 constructed in accordance with the invention. This matrix is provided with a plurality of equally spaced fins 12, the outer edges 14 of which define a substantially cylindrical plume which fits in a test tube 16, the outer edges 14 being located very close to the inner wall 18 of the tube. The test tube 16 functions as a container for the sample to be analyzed (quantitative or indicative determination of such components as antibodies and antigens). The die has a vertical rod-shaped handle 22, to which the radially projecting fins 12 are attached.

The radius of the fins is equal to or slightly less than the radius of the test tube 16. The bottom part 26 of each fin is preferably shaped in such a way that the matrix follows the bottom of the tube. The fins preferably form parts of a uniform structure comprising the central rod.

However, the fins can also be attached to the rod by any suitable joining technique, such as tongue and groove, or by means of any suitable adhesive.

The embodiment shown in Fig. 1 is provided with eight radially projecting and equally spaced fins, but a different number of fins and a different configuration can also be used. An increase in the area of the matrix by increasing the number of fins has been found to increase the rate of reaction between the moving and the fixed component. Furthermore, it has surprisingly been found that an matrix provided with eight fins in many cases gives a proportionally greater reaction rate in relation to a coated tube than can be explained by the difference in reactive area (see Figs. 7 446 666 _ 19 and 10). ). Similar advantages have been observed with a larger number of fins, such as 12, 16 and 18. In view of these observations, the preferred embodiment of such a matrix as shown in Fig. 1 is provided with 8-18 fins. An array of 18 fins is the most straightforward embodiment. When using such fin-equipped matrices, the number of fins is in practice limited by the increased difficulty in obtaining free drainage due to capillary adhesion of the reaction liquid, especially near the handle where the fins approach each other at an ever smaller angle with increasing number of fins.

A comparison between the results shown in Fig. 7 with an array of 8 fins and a coated tube indicates that factors other than the size of the exposed area coated with fixed component also affect the reaction rate. These factors are currently unknown, but it is assumed that the reduced average diffusion distance when using an 8-fin matrix (compared to using a coated tube) is one such factor.

Fig. 2 shows an embodiment which is identical to that shown in Fig. 1 in all respects except that it is provided with a plurality of holes 28 in the fins for increasing the area of fixed component wetted by the sample being analyzed. The holes 28 can extend completely through the fins. As shown in Fig. 2, the holes may be circular or elongated; they can also have any other shape. In order for the area of the matrix to be increased by making circular holes, the radius of the hole must be less than double the wall thickness 30.

Fig. 3 shows an embodiment comprising a handle 50, to which a flat disc 52 with a diameter slightly smaller than the diameter of the test tube, in which the matrix is intended to fit, is attached. A plurality of downwardly directed pins 54 are attached to the disc 52. The pins 54 may be the same length, or they may be of different lengths so as to follow the rounded bottom of the test tube. The pins shown in Fig. 5 have a non-circular cross-section, but other cross-sections are also possible. The pins 54 can be individually attachable or detachable. The embodiment shown in Fig. 3 is particularly suitable for complex analyzes involving several different test reactions in the same sample, but it is also quite generally useful in solid phase reactions.

Fig. 4 shows an embodiment with a central rod 22, to which a plurality of concentric cylindrical surfaces are fastened 446 666 by means of radially projecting struts 64. Fig. 4 shows two cylindrical surfaces, 60 and 62. In order to to accommodate the rounded bottom of the test tube, the inner cylinder 60 extends below the outer cylinder 62, and the central rod 22 extends further down than the inner cylinder 60. A group of lower struts 66 provide rigidity to the lower part of the die and facilitate also the drainage of liquid: Fig. 5 shows an embodiment with a radially projecting surface in the form of a spiral ramp 56, which winds around a central core 32 attached to a handle 22. This device comprises a continuous, substantially horizontal surface 36 and a vertical surface 54 formed by the ramp and by its outside, respectively. The slope of the ramp provides free gravity drainage of the reaction liquid when the device is taken up therefrom. The matrix in Fig. 5 is particularly suitable for carrying out reactions in which stirring is desirable. The matrix is tapered at the lower end 38 so that it fits in a test tube with a rounded bottom.

Fig. 6 shows an embodiment with a plurality of horizontally arranged discs 42 attached to a central handle 22. The discs 42 have substantially parallel upper and lower surfaces 44 and vertical edges 46. In the lower part 48 of the matrix a disc has a contour fitting to a test tube with a rounded bottom. Although the matrix shown in Fig. 6 is useful, it and similar embodiments lacking freely drainable surfaces require a longer washing time than the freely drainable embodiments of Figs. 1-5. Matrices with other geometric configurations than those shown in Figures 1-6 are also useful according to the invention. The geometric configurations should preferably have vertically oriented smooth surfaces in contact with the liquid sample to facilitate gravity drainage after being taken out of the test tube. According to the invention, any geometric configuration can be used which has a freely drainable, relatively large area. The larger the wettable area, the faster the reaction between the moving and the fixed component.

Before the insertion matrix according to the invention is immersed in a liquid sample containing a moving component, which is to be determined quantitatively or demonstrated qualitatively, all surfaces are coated with the fixed component. A large number of matrices can be coated 446 666 21 at the same time, whereby the manufacturing costs can be reduced to the point where it becomes economical to dispose of used matrices.

The matrices can be made of almost any stable, water-insoluble material, e.g. polymethacrylate, polypropylene and polystyrene.

Fig. 14a is a photomicrograph of the surface of a preferred embodiment, an 18-fin matrix formed in a polished mold. Magnification 500 times; electron beam scanning angle 450.

Fig. Lüb is a photomicrograph of the surface of a prototype matrix formed of the same plastic material as above (Fig. 14a) but in an unpolished form. Magnification 300 times; electron beam scanning angle 450.

The invention is illustrated by the following examples.

Example 1

Matrices of polymethacrylate were prepared in the form of rods provided with 2, 4 and 4, respectively. 8 fenor. In addition, a coated polystyrene pipe was prepared for comparison. The matrices were similar to the matrix shown in Fig. 1 except that the fins were not rounded in the lower part and therefore did not extend all the way down to the rounded bottom part of the reaction tube. The measured reactive surfaces were as follows.

Coated pipe 899 mmz Matrix with 2 fins 452 mm2 "" Ä "660 mm2" "8" 1075 m? The test reaction was an antigen-antibody reaction between 125 I-thyroxine (T4) and rabbit anti-thyroxine antibodies. The antibody was used as a fixed component, and the radioactive antigen was used as a mobile component.

To immobilize. the antibody on the above solid surfaces is diluted with a 2.5% solution of glutaraldehyde with an equal volume of 0.5 M sodium carbonate buffer pH 9.5. The solid surfaces were immersed in the glutaraldehyde solution for at least 3 hours at room temperature and then washed with water. The glutaraldehyde-treated surfaces were then reacted immediately with a 1000 dilution of anti-T4 serum in 0.5 M sodium carbonate buffer (pH 9.5) for 12 hours at room temperature.

The surfaces were then washed with water and stored in phosphate buffered saline consisting of 0.006 M NaH 2 PO 3, 0.024 M KEHPO 4 and 0.15 M sodium chloride (pH 7.4); to this solution had been added 446 666 22 0.1% bovine serum albumin and sufficient merthiolate to release any T4 bound to serum protein. The whole mixture is hereinafter referred to as T4 buffer.

Reactions were performed by incubating the coated surface carrying immobilized antibodyxin antibodies with a small amount of 125 I-thyroxine in a total reaction volume of 1.6 ml. The samples were incubated for various lengths of time at room temperature. The results are given in the table below.

Percent labeling substance bound by levitris with 2 fins 9.5 17.4 21.7 26.7 31.9 52.0 68.2 raatris with 4 fins 14.2 22.2 28.3 34.2 38.1 70, 6 75.7 Matrix with S fins 20.4 31.1 '40.3 36.6 58.7 81.4 85.3 coated tube 16.1 21.8 24.4 28.5 36.4 60.6 75.6 15 min 30 min 45 min lh 2 h l6h 65h The advantages of the fin-equipped matrices are clear from these results. It should be noted that the purity of the 1251 thyroquine used was 65-90%, so the maximum achievable degree of biting was 85-90%. Thus, when using the matrix with 8 fins, one had almost reached equilibrium after 16 hours. It is noteworthy that the matrix with 4 fins, the surface of which was only about 3/4 of the surface of the coated tube, immobilized antigen at a slightly higher rate than the coated tube. The matrix with two fins also immobilized more antigen per unit area than the coated tube. w hxemgel 2.

The same experiment was carried out in the same manner as in Example 1, the reactions being allowed to take place for different lengths of time at room temperature resp. at 37 ° C. The results at 3700 are shown graphically in Fig. 10. Fig. 10 shows that the matrix with four fins at room temperature bound a larger percentage of antigen than the coated tube for most of the reaction time even though it had a smaller area than the tube. At 37 ° C, the matrix with four fins bound no more antigen than the coated tube. In contrast, the matrix with 8 fins bound almost 1.6 times as much antigen as the coated tube after 60 minutes of incubation, even though its surface area was only 1.2 times the surface of the coated tube. 446 666 23 Example 2.

The behavior of different matrices was compared when increasing the antigen concentration. The experiments were performed in much the same way as in Example 1, except that the antigen concentration was varied by adding protein-based standards of unlabeled thyroxine, while keeping the concentration of 1251-thyroxine constant.

The antigen concentration varied from 25 to N00 nanograms of thyroxine per ml of serum, based on a sample volume of 0.025 ml. Matrices with 2, 4 and 8 fins were compared with a coated tube when incubated for 3 hours at 57 ° C in a total batch volume of 1.725 ml. The reaction buffer was the TO buffer described in Example 1. The results are shown graphically in Fig. 11. The fin-provided matrices according to the invention behaved in approximately the same way (curves of the same shape), while the coated tube behaved differently, which shows that the reaction rate is different in fin-provided matrices and coated tubes. It can also be seen that the curves of the fin-provided matrices (especially those with 4 and 5 fins) have a larger slope over a wider range of antigen concentrations than the curve of the coated tube has. The fin-plated matrices can therefore provide a better differential response over a wider range of antigen concentrations. Fig. 8 shows results from a similar experiment comparing a matrix of 5 fins with a matrix of 16 fins. In this experiment, the matrices were incubated for 1.5 hours at room temperature in a total reaction volume of 1.25 ml (ßfenor) resp. 1.025 ml (16 fins). The matrix with 16 fins is most suitable for quantitative analyzes, as it gives a larger response and a steeper response curve at most antigen concentrations.

Fig. 9 shows results from a similar experiment, using a matrix with 8 fins and carrying out a reaction at 22 ° C for 45 minutes. The total reaction volume was 1.525 ml, and the sample volume was 0.025 ml. Under these conditions, the 8-fin matrix gave a useful response throughout the concentration range tested, and the reaction was relatively rapid.

Example 4.

An array of 8 fins was compared with a coated tube in immunochemical determination of insulin. Insulin antiserum from guinea pigs was immobilized on the surfaces of tubes and matrices in the same manner as described in Example 1. The reaction was carried out in the same manner as in Example 1 except that insulin was used as the antigen. Each reaction medium contained an identical amount of 125 I-near insulin together with an amount of unlabeled insulin in phosphate buffered saline containing bovine serum albumin. The total reaction volume in both cases was 1.5 ml containing 500 μl of unlabeled insulin standard and 1 ml of phosphate buffered saline (see Example 1) containing 50 mg of bovine serum albumin per ml. The table below shows the experimental results as a percentage of bound labeling substance.

Percent bound labeled antigen 6.25 micron units 25 micron units 200 micron units' matrix coated matrix coated matrix coated Time, n acc. pipes according to pipes according to tube opg. opg. opg. l 35 12 33 12 18 6 2 45 19 53 12+ 25 8 3 51 20 44 16 30 7 The percentage degree of bonding was usually more than twice as large when using the matrix according to the invention with 8 fins as when using the coated tube although the surface of the matrix was only about 1.2 times the surface of the tube (see Example 1).

Examples Matrices were compared with 8 fins with coated tubes and the effect of different specific surface area, lack of fins extending into the rounded bottom of the reaction tube, and possible differences in bonding affinity between polymethacrylate and polystyrene were investigated. The reaction was carried out in the same manner as in Example 3, incubating at room temperature for a certain time. Both the tubes and the matrices were made of polymethacrylate. The tubes had a flat bottom, and the tubes and matrices had an equally large coated surface. The experimental results are shown graphically in Fig. 13. The results show that elimination of the above variables did not significantly change the general result in the previous examples, ie. that the bonding takes place faster with the fin-provided matrices according to the invention than with coated tubes even when the coated surface is the same size. Example 6.

Fig. 12 shows the results of a sandwich analysis for the determination of u-fetoprotein, wherein matrices with 8 resp. l2 fins were compared with a coated pipe. The antibody to α-fetoprotein was immobilized by adsorption. The fin-plated matrices were immersed in 1.5 ml of a solution containing 18 pg of α-fetoprotein antibody per ml of 0.5 M sodium carbonate buffer (pH 9.5) and incubated for two hours at 3700. Polystyrene tubes were similarly coated by 5 ml of the same antibody solution was introduced into each tube, then incubated under the same conditions. At the end of the incubation period, the tubes and matrices were rinsed, after which they were placed for 1 hour in phosphate buffered saline (pH 7.4) containing 10 mg of bovine serum albumin per ml. The tubes and matrices were then rinsed again, and assay buffer was added to the tubes containing the matrices with 8 fins (total volume 1.5 ml), to the tubes containing the matrices with 12 fins (total volume 1.1 ml) and to the coated tubes ( total volume 1.5 ml). The assay buffer was identical to the T4 buffer described in Example 1 except that it did not contain merthiolate and that the oxerum albumin content was 10 mg / ml. Alpha-fetoprotein was added to the reaction tubes in such amounts as to obtain final concentrations of 1 ng, 2 ng, 20 ng, 200 ng and 400 ng per mL of serum, based on a sample volume of 0.1 ml.

Immediately after the addition of d-fetoprotein, 100 ml of 1251-labeled antibody top was added to α-fetoprotein (total activity: 40,000 records per minute). The mixture was incubated at 7 ° C for 1 hour 15 minutes. After the incubation, the matrices were removed, and the tubes were decanted. Both the tubes and the matrices were quickly rinsed, after which the bound radioactivity was counted in a gamma counter.

From the results in Fig. 12 it appears that the fin-provided matrices according to the invention gave a greater differential response than the coated tube at all antigen concentrations, which is of particular importance at the lower concentrations. The matrix with 12 fins gave the largest response at all antigen concentrations tested and also gave a slightly larger differential response than the matrix * with 8 fins.

Example Z.

An immunoassay for the determination of thyroxine-stimulating hormone (TSH) was performed using a preferred embodiment of the invention. Polypropylene matrices of the type shown in Fig. 1 but with 18 fins were used. The matrices were prepared by molding in a mold with polished surfaces so that the fins attached to the handle obtained maximum surface smoothness.

The matrix was coated with anti-TSH antibody by immersing the finely divided portion in 1.2 ml of a solution containing about 1 ng of purified antibody and 10 ng of bovine serum albumin in 0.01 M potassium phosphate buffer (pH 9.0) for 2 hours. at BTOC. The matrix was then washed with water and immersed in a solution of gluaraldehyde (0.005% by volume) in 0.01 M potassium phosphate buffer (pH 9.0) for 2 hours at §7 ° C. The matrix was then washed with water, then washed for 10 minutes with a slow-flowing aqueous solution of "Triton X-100" 2 mg / ml; pH 1.85), washed again with water and stored until use in a phosphate buffered saline at 4%.

Matrices prepared in this way were immersed in tubes containing 0.2 ml of TSH standard (varying activity from tube to tube) and 0.2 ml of 1251-labeled anti-TSH antibody (total activity: l74 535 registrations per minute per tube ). A reaction of this type, in which the moving component and the indicator are incubated-simultaneously with the fixed component, is called a simultaneous sandwich analysis. One group of matrices was incubated for 2 hours at 5700, a second group was incubated for 3 hours at 5700. The total reaction volume was 0.4 ml.

The results obtained are summarized in the table below. 446 666 vi 2 m fi å SSS ä.

Sån ä SSS S2 2.2 SEN ä fi SSS 258. 3%: Sn 2.2 S22: Um H22 022 .SSS 3 Siw 82 Sá SSS Så SS 92 SSS 3.3 SNS 2% SS 22 m. .Så Son S ~ SSS än ä; ZS S4 S3 Qï fl SS 34 22. . š fl 33 RS S o S2: äó S3 83. S2 S Qßmp fi: wmo fi n fi uum mohx fi u ñvwu fl c _. -mm: mm »mm ^ mnw> w -fl wämmm ._ He pdnmw fi mnoxv: nswwmc fl sxnmë mmm nomsw fi wwmwm | n fi mmmcww ~ mmwwm .Hwuw w ^ \, wnmuwnsmwmc fi f fi: wøox mfl nc. .. n: omwïë ».šmw fi: mass \ .Hmmc..nmwn» w fl ww «a -wmn flfl xsms cwøc fl n. ïämwzwnmö psm fl s mms ubwvoäïwwwwm .ao fl gsxs fi s n Éwoopa fl wuwwh .co fi wnsmwm fi nww fi 446 666 28 The results of the duplicate experiments show that the reaction system is highly reproducible. The relatively insignificant increase in the amount of labeled substance in an increase in the incubation time from 2 to 3 hours shows that the reaction is rapid; the main part of the binding takes place already during the first two hours.

The system has such sensitivity that it is possible to measure TSH levels as low as 1.5 microunits per ml, while at the same time the capacity is sufficient for measuring the TSH content up to at least 100 microunits per ml.

Example 8.

A competitive type analysis was performed with a matrix of 18 fins. The matrix was coated with anti-digoxin antibody according to a method substantially similar to that described in Example 7. The amount of antibody applied was about 250 ng per matrix.

A reaction liquid containing 0.5 or 6.0 ng of unlabeled digoxin per ml and 1.3 H 3 digoxin (total 16 l; registrations per minute) in 0.01 M potassium phosphate buffer (pH 7.5E) was used. The total reaction volume was 1.2 ml. The matrix was incubated in the reaction vessel at 37 DEG C. or at room temperature for the time periods indicated in the table below. in the table, proeent bound labeled digoxin indicates (mean values of two determinationsš.

Reaction temperature Incubation time Digoxin concentration fng / ml- (minutes) 0 0.5 6.0 37 ° C 10 41.09.! 30, 3:15 5, 5% 30 å sno fi š 1 + 1,1: «ä 7,91% 90 1 65,2? 43.1% 7.9? Room temp. At 33.5% 26.6% 6.55 I 90 58.5% 43.7% 6.1% At the level of 0.5 ng / ml a maximum difference was obtained at an incubation time of 90 minutes at 5700, but fully measurable differences were obtained already after 10 minutes of incubation at both room temperature and 3700. 446 666 29 Exemnel Q.

A simultaneous sandwich analysis was performed to determine TSH in the manner described in Example 7, using a matrix with 6 parallel fins and a flat bottom (this embodiment is not shown in the drawing). This matrix was formed in an unpolished form (see Example 10).

TSH antibody was immobilized by adsorption (1.5 hours at 3700) in 0.01 M potassium phosphate buffer (pH 9.0) containing 1.5 μg anti-TSH antibody protein per ml and 15 μg 1 g G per ml. After the adsorption period, the matrices were washed twice with phosphate buffered saline containing 10 ug of bovine serum albumin per ml.

Reactions were performed in the presence of the amounts of TSH antibody listed below as indicator reagent (total activity: 127,000 registrations per minute). The reaction buffer was phosphate buffered saline, and the reactions were performed at 37 ° C for the time periods given below. To facilitate drainage and washing of the matrices after uptake of the reaction liquid, each matrix was quickly dried by placing the flat bottom against an absorbent pad, after which the matrix was centrifuged in a tube containing glass beads supporting the matrix so that the last traces of reaction liquid were removed. The matrix was then immersed in a wash buffer, and the process was repeated twice. The results obtained are shown in the table below.

TSH (microunits / ml) Percent bound label (corrected for 75% antibody affinity, 1 h 2 h 16 h O 2.2% 2.6% 4.4% 1 2.7% 4.4% 25 4, ¶% 5 .13 50 5.8% 7.5% 7.2% Although the matrix used is one of the least preferred embodiments, it also gives a very satisfactory result.

An array of 18 fins of the type used in Examples 7 and 8 was examined in a scanning electron microscope to determine the effect of the polished shape on the surface smoothness used in the manufacture. For comparison, the surface of a prototype matrix formed from the same plastic material in an unpolished form was also examined. Fig. 14a shows the surface of the matrix produced in the polished mold (bead B00 times; scanning angle 450). Fig. Lüb shows the surface of the matrix produced in the unpolished form (same magnification and scanning angle). The use of a mold with mirror-polished surfaces thus provides a greatly improved surface smoothness. Although both surfaces feel approximately equally smooth and consequently are within the scope of the invention, the matrix produced in the polished fornæn is usually preferred.

When using a preferred matrix, such as that shown in Fig. 1a, very reproducible results are obtained with a low background of non-specific binding.

Example ll. The matrix described in Example 7 was used in enzyme-linked immunoassay to determine ferritin, and the results were compared with those obtained using a coated tube. The assay was a sandwich assay, in which the matrix with 18 fins or the coated tube was coated with ferritin antibody and then reacted with a reaction liquid containing a ferritin sample. The matrix or coated tube was then separated from the reaction liquid, washed and incubated with an anti-ferritin antibody conjugated to an enzyme. Alternatively, a co-sandwich assay was performed, in which ferritin and anufferritin-antibody-enzyme-conjugate were incubated together down the immobilized anti-ferritin antibody. In both cases, binding of the islet antibody-enzyme conjugate to the solid phase was obtained in an amount proportional to the amount of ferritin present. The amount of bound antibody-enzyme conjugate was measured by the introduction of a chromogenic enzyme substrate, the bound enzyme portion of the conjugate giving a color change. The degree of color change was proportional to the amount of bound conjugate.

At the same incubation time, use of the 18-fin matrix gave an optical density of the order of 8 or more times the optical density obtained when using the coated tube.

Preferred embodiments of the invention have been described above, but it is obvious that many different modifications can be made within the scope of the invention.

Claims (9)

446 666 '31 P a t e n t k r a V 1. A method for determining a moving component in a liquid sample inserted into a container, wherein the moving component reacts with an immobilized component on a handle, submerged inside the liquid sample, characterized in that the immobilized component used has been applied in the form of a fixed uniform coating of at least 8, preferably 8-18 from the handle of the insert radially projecting fins with different surfaces and with such large dimensions in relation to the inner space of the container that the average diffusion distance of the molecules of the moving component to the phenyls coated with the immobilized component becomes considerably smaller than the average diffusion distance to the inner surfaces of the container in the absence of the insert; and measuring a chemical, enzymatic or immunochemical change caused by the reaction of the components on these phenytes as a measure of the concentration of the moving component in the sample. A method according to claim 1, characterized in that the immobilized component applied to the phenytes is an antibody, that the mobile component is an antigen, and that the reaction is binding of the antigen to the antibody. 3. A method according to claim 1, characterized in that the immobilized component applied to the phenytes is an enzyme, and that the mobile component is a substance which can undergo a chemical transformation catalyzed by the enzyme. 4. A method according to claim 1, characterized in that the mobile component is an enzyme, and that the immobilized component applied to the phenytes is a substance which can undergo a chemical transformation catalyzed by the enzyme. Device for determining a moving component in a liquid sample, comprising a container (16) for the liquid sample, preferably a test tube, and a handle-provided insert (10) immersable in the liquid sample in the container, carrying an immobilized component, intended to react with the movable component, characterized in that the insert (10) in determining the concentration of the movable component comprises at least 8, preferably 8-18 from the handle of the insert to different holes1 \ fi $ projecting fins (12), which has smooth surfaces, the immobilized component in the form of a fixed uniform coating I 446 666 32 and has such large dimensions in relation to the internal space of the container (16) that the average diffusion distance of the molecules of the moving component to those with the immobilized component coated phenytes become considerably smaller than the average diffusion distance to the inner surfaces of the container in the absence of the insert. Device according to claim 5, characterized in that the outer edges of the fins (12) are formed in accordance with the shape of the container (16). Device according to claim 5 or 6, characterized in that the insert (10) comprises 16-18 fins (12). Device according to one of Claims 5 to 7, characterized in that the insert (10) is manufactured by molding in a mold with polished surfaces. Device according to any one of claims 5-8, characterized in that the handle (22) is a central rod, the fins (12) extending along a part of the length of the rod.