NO861229L - PROCEDURE AND TESTING DEVICE FOR DETERMINING ANALYSTS. - Google Patents

Description

The invention relates to a method for assessing agglutinations caused by complexation of bioaffinity binding partners using a signal-producing system. The invention further relates to a test device with the help of which the method is carried out.

The predominant number of binding reactions between bioaffinity binding partners leads to a complex, which has a greater spatial extension than each of the individual binding partners. This change in structure is particularly pronounced when a binding partner of the bioaffinous binding pair from nature is particulate or is linked to a dispersible particle or to a large soluble polymer molecule. The particles present as monomers before the binding reaction form dimeric or oligomeric aggregates during the binding reaction. Examples of this are bacterial egg agglutination, bentonite agglutination, hemagglutination and latex agglutination.

In an analogous manner, a latex agglutination inhibition test for the determination of haptens, e.g. penicillin, a separation between immunochemically aggregated and non-aggregated latex particles. Thereby, sensitized latex is mixed in a syringe with a penicillin-containing sample, incubated and pressed under pressure through a pre-moistened filtration membrane and the non-aggregated part is determined in a spectrophotometer using the light scattering property.

Unfortunate with this method is the number of operations

which is requested by the user, such as e.g. dosing and mixing of reagents and samples, exact observance of incubation times, pressure filtration, collection of the filtrate, determination of the proportion of aggregated and non-aggregated particles. Alongside the corresponding manual work, there is also the possibility of operating errors,

which falsifies the result of the determination procedure.

In particular, the separation of the aggregated and non-aggregated particles on a filter membrane in a reproducible manner is very difficult, as depending on the applied pressure there is an enrichment of particles in the vicinity of the membrane which decisively affects the filtration ratio. Correspondingly, at different times of the filtration process, different amounts of non-aggregated particles pass the membrane, so that extraction of a representative amount of filtrate for the subsequent determination procedure is made difficult.

A method was found, by which the agglutinates of particles produced by bioaffinity interaction are separated from the dispersed particles by independent filtration according to the type of chromatography, and the part of dispersed particles is detected using a signal-producing system (SPS). The procedure is carried out using a device which enables independent filtration according to the type of chromatography.

The object of the invention is a method for detecting and determining the concentration of an analyte present in aqueous solution with bioaffinity binding properties by agglutination or by agglutination inhibition of particles, which are equipped with bioaffinity binding properties and at the same time with an SPS or with parts of an SPS, as the method is characterized by

a) a separation of agglutinated and non-agglutinated particles is carried out using a device comprising

of materials which absorb and transport aqueous solutions and particles dispersed therein, and b) the separated part of non-agglutinated particles be-. voted on its SPS.

The particles used in the method also contain, in addition to the components, which give them their bioaffinity binding properties, at least one component of SPS. The method is carried out with the help of a device which, according to the type of chromatography, enables the independent separation of agglutinated and dispersed particles using materials which absorb and transport aqueous dispersions, but retain agglutinates. Thereby, the solution containing the analyte is applied to a

of the area of the agent designed as application or reaction zones. The analyte reacts either with the particles contained in the reaction zone in solid form or applied as a dispersion to the separation zone and possibly with the other previously mentioned components of the bioaffinity binding system. The agglutinates that form were retained on or in an area of the agent designed as a separation zone. The particles remaining as a dispersion travel with the liquid through the separation zone and enter an area designed as a detection zone where, under the influence of all components of the SPS, they produce a measurable signal.

The object of the invention is further a device for detecting and determining the concentration of an analyte present in aqueous solution with bioaffinity binding properties by means of particles in an agglutination reaction, whereby agglutinated and non-agglutinated parts are separated from each other, the device being characterized by

a) it contains an application, a reaction, a separation and a detection zone, b) all zones consist of materials, which absorb and transport aqueous liquids, c) all said zones are permeable to the non-agglutinated particles, d) in the smallest separation zone is impermeable to agglutinated particles, e). the device contains a signal-producing system, which produces an evaluable signal in the detection zone.

The device can consist of several flat-shaped structures or small plates, which are in contact with each other or can be brought into contact, touching at their surfaces of smallest extent, are arranged next to each other, i.e. flat-shaped or touching at their surfaces of greatest extension, i.e. layered. A combination of surface and layer-shaped device is also possible. The small plates can be attached to holders or to carriers in the mentioned devices. The small plates consist of materials that absorb and transport aqueous solutions and particles dispersed therein.

When the device, for example, consists of three plates,

these designed as a reaction, separation and detection zone, it consists of four small plates, then the extra plate is designed as an application zone.

Exemplary embodiments of the device are shown in longitudinal section in Figures 1, 2, 3 and 4. The holders or carriers are denoted by A. They consist of flat-shaped structures of plates or strips made of materials that do not absorb aqueous solutions. At it on

fig. In the device shown in 1, the carrier is a transparent plate. At those in fig. 2, 3 and 4 shown devices are the carrier

not transparent, but equipped with recesses or slits a. E denotes the plates designed as application,

B those designed as reaction zones, C those designed as separation zones and C those designed as detection zones. In the devices according to fig. 2 and 3, the holder consists of two wing-shaped strips or plates, which are connected to each other at an edge respectively and which are folded over each other during use. One wing contains on its upper side in a flat arrangement, i.e. lying next to each other application zone E and reaction zone B, the other wing in a layer-shaped arrangement on its underside and in the recess extending upwards the separation zone C, on its upper side and in the recess extending downwards into the detection zone D, touching the separation zone C. Fig. 2 and 3 are separated by different large application resp. reaction zones E or B.

Fig. 4 shows a device where, on the inside of the carrier A, the application and reaction zones E and B are arranged flat next to each other. Through the recess a', the application zone E is available for taking up samples and possibly for taking up additional solutions. In the recess a, the separation zone c is incorporated and above that the detection zone D is placed on the upper side of the carrier A touching the separation zone C with the underside.

In the case of the presence of an application zone E, it serves to absorb the solution of the analyte. It can also serve to absorb a solution with components of SPS and/or a dispersion of particles for the agglutination reaction as well as an additional eluent.

The reaction zone B serves for formation or inhibition

of the agglutination of particles with the participation of all components of the bioaffine binding system as well as possibly for the uptake of a dispersion of particles and of the dissolution of the analyte, when no uptake zone E is present. It can also contain the particles as well as the soluble components of the bioaffine binding system and of SPS in dry form.

The separation zones C serve to separate agglutinated particles that occur in the reaction zone. The agglutinates were either retained at the interface of the reaction and separation zone or taken up by it and retained. It is permeable to non-agglutinated particles and to all other soluble components that are necessary for the determination of the analyte. The separation zone can be made from materials with fibrous as well as spherical structure, i.e. from materials with anisotropic as well as isotropic porous structures.

The detection zone D is used for recording particles that have migrated and become dispersed after the bioaffinity reaction has taken place and their detection using SPS. It is preferably made from materials that have a high degree of remission for radiated light. If, for example, the signaling unit is a chromophore or fluorophore, then no further components are required for SPS, the detection takes place by visual assessment, possibly by means of a color comparison scale or by

■ref lex ionsf otometry or ^f luorometry . In the preferred embodiment of the determination method, in which the signaling unit is an enzyme, due to the achievable sensitivity, the enzyme activity of the part of the SPS that passes the separation zone is determined in the detection zone. This can take place by adding an enzyme substrate solution to the detection zone, preferably, however, the detection zone contains all components of the enzyme-substrate reaction. With certain enzymes, e.g. peroxidase, it can of

reasons for reagent stability, it may be necessary to produce the detection zone from two zones, which are connected to each other by suction and in which respectively one or more components of the substrate reaction are incorporated separately from the others.

As particles, latexes with a hydrophilic surface are preferred, especially those with a core-shell structure, as they re-

are mentioned in DOS 31 45 082. Other particles dispersible in aqueous solutions are also suitable. To the particles, at least a component of that for the analyte is specific

obtained bioaffinity spinning systems and at least one component of SPS bound. Covalent bonds are preferred for the bioaffinity binding partners and for the components of SPS,

but also the avidin/streptavidin-biotin systems are suitable for binding the components of SPS to the particles.

Bound components of SPS can be enzymes such as glucose oxidase, alkaline phosphatase, peroxidase or beta-galactosidase as well as chromophores or fluorophores.

The partners or components of the bioaffinity system which, in the presence of the analyte, contribute to the agglutination of the particles or to their inhibition, insofar as it does not concern the binding partners bound to the particles, are soluble components which are placed in the device in solid form or can be applied in a resolution of the previously mentioned application or reaction zone.

An agglutination takes place when, for example,

a) the dispersed particles contain at least one biofunctional bioaffinity binding partner of the analyte and b) the dispersed particles contain the analyte or a binding-capable derivative of the analyte and at least

bifunctional binding partner of the analyte is present.

An inhibition of the agglutination by the analyte takes place when the dispersed particles contain at least one bifunctional binding partner of the analyte and one at least bifunctional derivative of the analyte is present.

The invention will be explained in more detail with the help of some examples.

Example_el_l

Preparation of a test device for the detection of antibodies against Streptolysin-0 (SL-O)

1.1. Latex particles with core-shell structure The production of latex particles suitable for covalent binding of proteins took place as described in example 2b of DE 31 45 082 using polystyrene latex cores. The latexes with core

shell structure obtained after a graft polymerization with styrene, methacrylic acid and methacrylamido-racedaldehyde di-n-pentyl acetal had a particle diameter of 160 nm,

the content of releasable aldehyde groups constituted

0.05 mval/g latex.

1.2 l-"peroxidasyl"-l,2,9,10-tetraza-3,8-dioxodecane

5 mg lyophilized sea radish peroxidase (POD), 250 U/mg

at 25°C (Boehringer Mannheim, purity grade I, order no. 121606) was dissolved in 2 ml of a buffer of 0.1 mol/l sodium hydrogen phosphate, pH 6.0, to which was added 0.5 ml of a freshly prepared solution of 50 mmol/1 sodium metaperiodate in water and incubated with stirring for 10 minutes at room temperature under light protection. The reaction mixture was then freed from periodate by gel filtration on "Sephadex" G-25, equilibrated with the above-mentioned phosphate buffer. The peroxidase-containing fraction of 3 ml was incubated with a solution of adipic acid dihydrazide at a final concentration of

0.1 mol/l 2 hours at room temperature in the presence of 1 g/l sodium cyanoborohydride. The mixture was then dialysed at +4°C for 2 days by several buffer exchanges against the above-mentioned phosphate buffer.

1.3 l-"phosphatacyl"-l,2,9,l0-tetraaza-3,8-dioxodecane \ 26 m<q>of a 1 V° f i 1 i s a. t of alkaline phosphatase of calf intestine (100 U/ mg lyophilisate corresponds to approx. 1300 U/mg protein at 37°C - Boehringer Mannheim, order no. 405639, purity grade I) was reacted analogously to example 1.2 with sodium metaperiodate and adipic acid dihydrazide with the change that said phosphate buffer had been adjusted to pH 7, 0 and the dialysis was carried out with a buffer of 50 mmol/l sodium borate, 10 mmol/l magnesium chloride, 0.1 mol/l zinc chloride, adjusted with caustic soda to pH 8.0.

1.4 Binding of l-"peroxidasyl"-l,2,9,10-tetraaza-3,8-dioxodecane and of Streptolysin-0 to latex.

10 ml of a 1% suspension of it according to example 1.1

prepared latex was mixed with 0.5 ml of 1 normal HCl and incubated for 15 minutes at room temperature. Then 0.4 ml of 1 normal NaOH, 1 ml of a saturated sodium hydrogen phosphate solution, pH 6.5, 8 ml of a 0.9% NaCl solution, 200 µl of the

in example 1.2 formed a solution of a 1-"peroxidasyl"-1,2,9,10-tetraaza-3,8-dioxodecane and 1 ml of a solution of 1 g/l sodium cyanoborohydride and incubated for 30 minutes at room temperature. That was it

put 0.4 ml of an aqueous solution of 10 g/l

Streptolysin-0 and 0.5 ml of an aqueous solution of 200 g/l polyoxyethylene sorbitan monolaurate, known under the trade name "Tween" 20 and incubated overnight at room temperature. Then 1 ml of a solution of 0.5 mol/l L-aspartic acid which had been adjusted to pH 6.5 with caustic soda and 0.5 ml of a solution of 1g/l sodium cyanoborohydride were added. It was incubated for 1 hour at room temperature, and the mixture centrifuged at approx. 50,000 x g. The residue was re-suspended in 20 ml of 50 mmol/l tris/HCl, pH 7.4 with the addition of 1 g/l human serum albumin. After repeated centrifugation, the residue was again taken up in 20 ml of the solution of tris/HCl, pH 7.4 with the addition of human serum albumin and homogenized using ultrasound.

1.5 Manufacture of a test device

A polyester foil, thickness 200 ym, width 8 cm, was used as an inert, water-impermeable plastic carrier. A double-sided adhesive tape (company Beiersdorf, Hamburg) with a width of 0.8 cm was respectively placed on the foil at a distance of 2 cm from the two side edges. This surface of the foil is thus declared as the front side. On one side, the back of the polyester foil was also equipped with the double-sided adhesive tape at an equal distance. In these strips of the foil covered on both sides by the adhesive tape, round holes of 0.5 cm diameter were then punched with a distance from hole center to hole center of 0.8 cm and on the remaining adhesive tape parts on the front of the polyester foil a 0.8 cm wide strip of a membrane filter, type RC-55, pore size 0.2 ym from the company Sartorius, Hamburg. A 0.5 cm wide strip of chromatography paper No. 1507 from the company Schleicher&Schuell, Dasssel was fixed on the side facing the center of the 2nd adhesive tape on this front, which formed the reaction zone and was previously moistened with a solution of 0.2 mg/ml tetramethylbenzidine in ethanol and prior to sticking had been dried using a ventilator. The backside adhesive tape was likewise stuck on with a 0.5 cm wide paper strip No. 1507, which had previously been moistened with a solution of 10 mmol/1 of an equimolar urea.H2O2 adduct in 50 mmol/1 citric acid, adjusted to pH 5.0 with a saturated solution of Na2HPO^, and had been dried (detection zone). With paper scissors, the foil web was cut crosswise to the glued paper webs in strips of 8 mm width.

1.6 Determination of anti-Streptolysin-0 (ASL-0)

The test element mentioned in example 1.5 was used for ASL-O determination. This detects antibodies against Streptolysin-O, a streptococcal antigen. In addition, a human immunoglobulin preparation from Behringwerke, which has the trade name "Beriglobin", was adjusted by dilution with phosphate-buffered physiological saline solution to a content of 300 IU/ml, 150 IU/ml, 75 IU/ml, etc. anti-Streptclysin-0 . 50 µl of this is respectively mixed on a black glass plate with 50 µl of the dispersion of latex mentioned under 1.4, to which Streptolysin-0 and POD were bound. This mixture was immediately divided: 50 µl was brought to the reaction zone of the test element mentioned in .1.5 and the remaining 50 µl moved by rotary tilting of the glass plate for 1 minute. The agglutination strength of the latex on the glass plate was assessed visually in the usual way from 0 - +4. The part applied to the sample device was incubated for 5 minutes in the reaction zone, then the strips were bent over and thereby the reaction, separation and detection zone were brought into acidic connection. The blue-green coloration formed in the detection zone after 5 minutes was determined by reflectance photometry using a rapid reflectance photometer from Behringwerke AG Marburg, a test strip evaluation apparatus.

The results obtained are listed in table 1.

Example_el_2

Production of a latex particle-containing test device for the determination of antibodies against Streptolysin-0 and use of this test device for the determination of the antibodies.

With the dispersion of latex prepared according to 1.4, to which peroxidase and Streptolysin-0 were bound, was

a glass plate moistened a paper machine wire with a mesh number of 225/cm 2 (the company Kufferrath, Reutlingen) in an amount of 20 ml/cm 2 and then lyophilized. Using a double-sided 0.8 cm wide adhesive tape (the company Beiersdorf, Hamburg), a 0.8 cm wide strip of the lyophilized latex-containing paper machine wire was attached to the reaction zone at the test device mentioned in 1.5.

On this surface, respectively, 50 µl of the anti-Streptolysin-0 dilution series mentioned under 1.6 was applied. After the liquid front had reached the end of the reaction zone, a bend formed the connection to the detection zone. The color formed in the detection zone after 15 minutes was assessed with the reflectance photometer already mentioned under 1.6. The result is reproduced in table 2.

Claims (14)

1. Method for detection and for determining the concentration of an analyte present in an aqueous solution with bioaffinity binding properties by agglutination or by agglutination inhibition of particles equipped with bioaffinity binding properties and at the same time with a signal producing system (SPS) or with parts of an SPS, characterized by that a) a separation of agglutinated and non-agglutinated particles is carried out using a device consisting of materials that absorb and transport the aqueous solutions and particles dispersed therein, and b) the separated fraction of non-agglutinated particles is determined above its SPS. 2. Method according to claim 1, characterized in that the solution of the analyte is applied to an area of the device which is equipped as a recording zone, the solution then traveling through areas which are equipped in the aforementioned order as a reaction, separation and detection zone. 3. Method according to claim 2, characterized by that a) that the analyte in the reaction zone produces or prevents an agglutination of particles that are equipped with bioaffinity binding properties and at the same time with an SPS or parts of a SPS, b) the non-agglutinated particles pass the separation zone and enter the detection zone, and c) the non-agglutinated particles in the detection zone are detected and determined by SPS. 4. Method according to claim 2, characterized in that particles dispersed in an aqueous solution with bioaffine binding properties are applied to the reaction zone at a time that is before the application of the solution containing the component to be applied to the application zone or coincides with this. 5. Method according to claim 2, characterized in that a device is used which in the application zone or in the reaction zone or distributed over both zones contains particles with bioaffine binding properties in dry form. 6. Method according to claim 2, characterized in that a device is used which in the detection zone contains components of an SPS which, together with the particles that have migrated to the detection zone, produce a measurable signal. 7. Method according to claim 2, characterized in that a device is used, on which the application and reaction zone are combined into a single area. 8. Device for detecting and determining the concentration of an analyte present in aqueous solution with bioaffinity binding properties using particles in an agglutination reaction, whereby agglutinated and non-agglutinated parts are separated from each other, characterized in that a) the device contains an application, a reaction, a separation and a detection zone, b) all zones consist of materials that absorb and transport aqueous liquids, c) all said zones are permeable to the non-agglutinated particles, d) at least the separation zone is impermeable to agglutinated particles, e) the device contains a signal-producing system,: which produces an appreciable signal in the detection zone. 9. Device according to claim 8, characterized in that it contains agglutinable particles in dry form. 10. Device according to claim 8, characterized by either a) a bioaffinous binding partner of the analyte and at least one component of SPS is bound to the particles, or b) a predetermined amount of the analyte or a derivative of the analyte and at least one component of SPS are bound to the particles, with at least one bifunctional binding partner of the analyte being present in the application zone or the reaction zone, or c) . a bioaffinous binding partner of the analyte and at least one component of SPS are bound to the particles, with at least a bifunctional derivative of the analyte being present in the application or reaction zone. 11. Device according to claim 8, characterized in that the zones touch each other with their surfaces of greatest expansion and are arranged in order below one another as an application, reaction, separation and detection zone. 12. Device according to claim 8, characterized in that it consists of two wing-shaped components which are attached to each other with one of their ends, so that they can be clapped together with their inner surfaces touching, one component containing the application and reaction zone next to each other, touching each other with the surface of smallest extent and the other component has a separation and detection zone in a device lying on top of each other touching each other with the surface of greatest extension. 13. Device according to claim 10, characterized in that the reaction and separation zone are arranged in such a way that when they overlap each other, they touch each other with respectively one of their surfaces of greatest expansion. 14. Device according to claim 8, characterized in that the application and reaction zone are arranged next to each other touching each other with respectively one of their surfaces of smallest expansion, that the separation zone comes to lie above the reaction zone, touching each other with respectively with one of their surfaces of greatest expansion and that the detection zone is arranged over the separation zone, touching each other with respectively one of their surfaces of greatest extension.