CS262653B2 - Process for performing immunochemical tests and device for making thereof - Google Patents

Abstract

The present invention relates to devices for carrying out quick immunochemical tests and to methods for the immunochemical determination of suitable proteins using this device. The device consists of a layer which has capillary activity and consists of a dry, porous, non-swelling, particulate material with molecular sieve properties, the pore size of the molecular sieve allowing penetration of the soluble antibodies used for the immunochemical detection of the antigens but allowing little or no penetration of the soluble immune complexes formed from antigen and antibodies. The layers with capillary activity are preferably contained in a glass or plastic capillary column or are attached to an inert support material. In the method according to the invention, a substrate sample is loaded onto one end of the device, and the immune complex which is formed where appropriate is separated from the antibodies by chromatography using a solvent and is detected.

Description

The present invention relates to a method for carrying out immunochemical rapid tests based on the use of chromatographic auxiliaries and apparatus for carrying out the method.

Immunochemical assays are becoming increasingly important for the determination of hormones, proteins, pharmaceuticals, etc., for example in blood, urine and faeces samples, but also outside clinical chemistry, for example in analytical food or wastewater analytical assays.

The advantage of these assays lies in the high specificity and affinity of the immuno reagents, which allows to safely determine even small amounts of substrates to be determined in addition to high concentrations of relatively similar compounds. The disadvantage here is that, especially for laymen, many handling steps, which are very time-consuming in conventional tests and can normally only be carried out with extensive laboratory equipment. For wider use of immunochemical assays, it is therefore desirable to have specific tests capable of determining lower amounts of the test substance which could be used without complicated additional equipment and preferably without additional pretreatment or post-treatment of the substrate and with an indication of the result in a short time.

It was therefore an object of the present invention to provide rapid tests by means of which immunological assays could be carried out easily and quickly by lay persons.

For example, the following reaction procedures are known for immunochemical assays:

1. The method of competitive custody

In this method, an accurately known amount of the corresponding labeled antigen is added to an unknown amount of the antigen or hapten (hereinafter referred to as the antigen), which is then reacted in competition with the corresponding antibody which is fixed on a solid support. The solid and liquid phases are separated and the amount of antigen to be determined is determined by determining the labeled antigen in the solid or liquid phase.

2. IEMA principle

Here, the assay antigen is reacted with the labeled antibody in excess, and in a second step, the unreacted antibody is reacted to the corresponding antigen fixed on the support. Again, after phase separation, the amount of antigen can be judged by determining the labeled antigen in solid or liquid phase.

3. The principle of sandwiches

In this method, the bivalent antigen is reacted with both the antibody bound to the solid phase and the labeled antibody and again after the liquid and solid phase are separated, the concentration of the labeled antibody is determined in one of the two phases and the concentration of the bivalent antigen is determined.

All of these methods have in common that one of the two reactants is bound to the carrier or is additionally bound to allow separation of the complex and excess reagent. Apart from the fact that this fixation to the carrier entails additional costs and, in particular, that the immunochemical properties may be altered, these methods require, as already mentioned, relatively high costs in carrying them out. For this reason, it is not suitable for simple, cheap methods of rapid tests.

For the rapid tests according to the invention, therefore, no carrier-bound or antigen-fixed antibodies should be used, but antibodies which fall directly upon immunization, or in their labeled form, should not be used. Antibodies according to the invention are to be understood as meaning whole antibodies but also active fragments of the antibodies, for example Fab fragments. The antibodies can be used in both polyclonal and monoclonal forms. Surprisingly, it has been found that this is possible because immunocomplexes from the antibody and antigen are allowed to be chromatographically separated due to their different molecular weight and / or different sizes from unreacted antibodies or antigen, and corresponding evidence in chromatographically separated fractions is then easy.

Methods of protein separation due to their different size are well known as gel filtration on, for example, Sephadex, Sephacryl and ultrogel columns. However, these methods have special requirements, so that they can only be performed by trained people with appropriate laboratory equipment. For example, the gel material must be pre-swollen for several days, the chromatographic columns must be carefully filled, equilibrated with the solvent, and always kept moist to achieve good separation.

In addition, solvent storage tanks, pumps, fraction collectors, detector systems, etc. are required in addition to the columns, and in addition, sample preparation time and chromate ography will require more hours, so these methods are not an option for carrying out rapid tests.

Surprisingly, it has now been found that a capillary-active layer consisting of a dry, porous, non-swellable granular material having molecular sieve properties (e.g., porous glass, silica gel, etc.) can be used to rapidly divide substrates of appropriate molecular size. based on capillary forces.

Depending on the pore diameter, the chromatography of the substrates proceeds faster or slower in accordance with their molecular sizes, with high molecular weight substances which, due to their size, cannot penetrate into the pores, are chromatographed immediately behind the solvent front. In this way, it is possible to carry out a corresponding division without relatively large loads, i.e. without prior equilibration of these columns or without additional pumping of liquids by pumps, in relatively short periods of time - 5 to 30 minutes at 5 to 20 centimeters. In order to avoid "oblique flow" of the columns, the diameter of the column should be 0.5 to 5 mm, preferably 1 to 2 mm. Since the immunocomplexes are usually substantially larger than the antibodies and antigens used to generate them, it is possible, using suitable molecular sieves, to choose a pore diameter so that the immunocomplexes either revolve at all or only a little, i.e., chromatographically along with or shortly after the solvent front, while the antibodies can penetrate into the pores and thereby be retained or chromatographed substantially slower. The foregoing dry capillary columns can therefore be used for immunoassay.

Accordingly, two different methods are possible according to the invention:

An antigen-containing substrate may be supplemented with an excess of antibody and preincubated. After the reaction has been carried out, the mixture is then loaded onto the molecular sieve described above and chromatographed either with a suitable solvent, i.e. normally an aqueous buffer solution, or with an excess of the reaction solution. The queue of solvent free of unreacted antibodies can then be determined by one of the known methods of immunocomplex concentration.

2. However, it is easier for the user to use prefabricated columns which contain a molecular sieve material impregnated with suitable antibodies in the lower part thereof and an unimpregnated chromatography material in the upper part. If a portion of the impregnated antibody is now coated with an antigen-containing substrate, an immunocomplex is formed in the mobile phase which, due to its size, cannot penetrate into the pores of the molecular sieve and thus travels faster than the unreacted antibody remaining in the stationary phase in the pores.

It seems extremely surprising that it is possible with such simple means to achieve a rapid and usable separation, as according to literature data, it would have to be expected that dry columns flowing through the liquid would only be imperfectly wetted, ie creating more or less large air inclusions. inside and between the particles, thus preventing separation. On the other hand, it was necessary to assume that especially in method 2, the formation of large immunocomplexes, which can only be located outside the pores, is so slowed by diffusion processes that a sufficient reaction rate does not occur within a short contact time of several seconds.

Accordingly, the present invention provides a method for carrying out immunochemical rapid tests in which antigens react with antibodies to form immunocomplexes, characterized in that the test substrate is deposited on the lower part of the apparatus and chromatographed by solvent elution with a layer, and the immunocomplex is formed. the top of the device.

In the method, the lower portion of the device is filled with an antigen-containing substrate and is introduced into a solvent, for example water or an aqueous buffer solution, which capillaryly rises up through the device and separates the antibody from any immunocomplexes formed.

The substrate solution containing the antigen serves as an eluent in chromatography.

Either the immunocomplex itself or its associated labeling enzyme is detected after chromatographic separation.

The apparatus for carrying out the method of the invention comprises a capillary active layer consisting of a dry, porous, non-swellable material that is grainy. The device according to the invention is characterized in that the antibodies, antigens and immunocomplexes are soluble compounds and the material exhibits molecular sieve properties, wherein the molecular sieve pores are larger than the antibody but smaller than the immunocomplexes formed from the antigen and the antibody.

The capillary active layer forms a capillary column. The capillary-active layer is deposited on an inert support, for example a plastic film or glass. The antibody is impregnated at the bottom of the molecular sieve.

Alternatively, the antibody is impregnated on the porous layer upstream of the capillary layer.

The antibody is labeled with a radioactive substance, a fluorescent substance or a chromophore.

Alternatively, the antibody is labeled with an enzyme. The top of the material is impregnated with a reagent system for the labeling enzyme.

The molecular sieve material comprises an inert, hydrophobic, fine-grained material such as talc, hydrophobized glass or silica gel.

The molecular sieve consists of a hydrophilic material, for example silica gel or porous glass, and / or is impregnated with a wetting agent, for example cationic, anionic or non-ionic, such as glycol ethers, alkyl sulfates, phenol ethers and bis-2-hydroxyethylamine lauric acid.

The molecular sieve material is additionally impregnated with buffers and / or salts and / or complexing agents or other active agents.

In a preferred embodiment of the invention, the capillary of glass or plastic, having an inner diameter of 0.5 to 5 mm, preferably 1 to 2 mm and a length of 5 to 20 cm, preferably 10 to 20 centimeters, is closed at one end by a wicking, loose material. A 0.2 to 10 mm thick layer of molecular sieve material impregnated with the labeled antibody is applied to this closure material and the column is filled with another unimpregnated molecular sieve and resealed at the top end with a permeable closure material. When the labeled antibody cannot be directly detected, for example due to its color, fluorescence or radioactivity, the top of the column may still contain an indicator area in which, for example, the corresponding reagents are contained to form a detectable product together with the labeled antibody, for example.

If several, µl of the test solution is injected into such a column at its lower end, and the column is then placed in a suitable developing liquid, then the liquid travels up to complete coverage of the column contents due to capillary forces upwards. In the presence of the antigen, as described above, a complex is formed and the complex is separated at the upper end of the capillary column. Since chromatography is automatically terminated by complete humidification, no special user control is required. The result can be read even after a long time, especially in a qualitative test, and possibly even evaporate the solvent and store the column for documentation purposes.

In a further preferred embodiment, the molecular sieve material is spread on a non-absorbent carrier in a layer of 0.05 to 1 mm thickness, optionally with addition of binder, on a non-absorbent support and is divided into strips 5 to 10 mm wide and 50 to 150 mm long In use, one end of these strips is impregnated with the corresponding antibodies and serves to sample application, and the other end is additionally impregnated with label reagents for labeled antibodies and serves to detect the formed antibody-antigen complexes. If these three areas are applied separately, it must be ensured that there is sufficient capillary contact between them. It is, however, also possible, first to apply, for example, a spatula, a coating material which is suspended in a suitable solvent, to dry it and then to impregnate with reagents.

Strips of plastic, for example polycarbonates, polyesters, polyamides or polyethylene, are preferably used as the impermeable support, but glass supports may also be used.

As mentioned above, the molecular sieves used for chromatography should consist of a non-swellable material. Accordingly, inorganic carriers and, in particular, various types of commercially available porous glass or silica gel with a defined pore size are suitable. The inner and outer surfaces of the inorganic carrier material may be coated with a suitable organic substance, for example a polyol, which imparts the adsorption properties of the agar or cellulosic gel and avoids the negative effects of the inorganic carrier on the antibodies or antigens.

Since aqueous solvents are mainly solvents for chromatography, the materials used must be sufficiently hydrophilic. If this is no longer guaranteed by the production of the materials, it has proven advantageous to suspend the carrier materials prior to processing in an aqueous solution containing wetting agents and a suitable buffer and to dry the impregnated beads again.

All known, anionic, cationic or nonionic surfactants can be used as wetting agents. Preferably, concentrations of 0.01 are used. up to 0.1%, but lower concentrations may be used, especially for materials that are naturally hydrophilic; higher concentrations may also be used as long as they do not affect immunocomplexes.

Wetting agents include, for example, glycol ethers such as polyoxyethylene stearate, polyoxyethylene laurate, alkyl sulfates such as dodecyl sulfate, polyglycol ethers of higher aliphatic alcohols, phenol ethers such as 10,5-ethoxynonylphenol, cetyltrimethylammonium bromide and bis-2-hydroxyethylamine.

The other components of the impregnating solution correspond approximately to those contained in the later developing solution, i.e. buffers, salts, complexing agents, sterilizing agents, etc., which, as low molecular weight components, chromatograph more slowly than the solvent queue, thereby ensuring that the solvent queue is stationary.

Furthermore, it has proven advantageous to admix the chromatographic material with an additionally finely powdered, inert and optionally hydrophobic filler, in particular talc, hydrophobized glass, silica gel and the like have proved to be advantageous. This material reduces the fill volume between the carrier particles. on the other hand, it slows the elution rate of the liquid phase thereby improving equilibrium due to improved diffusion between the mobile and stationary phases, and again improving the separation performance. In addition, the column packing is more homogeneous, thereby additionally preventing the formation of air inclusions.

When choosing binders for the production of coated carriers, care must be taken that, on the one hand, it results in stable coatings but, on the other hand, does not substantially alter the elution rates of the carrier materials, i.e., in particular, the pores are not reduced. This is surprisingly achieved by the use of an aqueous dispersion of plastic, for example polyvinyl propionate or polyacrylate, in admixture with a well water-soluble or swellable binder. Agar -? 9 was used with success

575 U / ml dissolved in 10 mM KP _b, 5 mM magnesium chloride, 25 mM sodium chloride NaCl, 2% sucrose, 0.5% bovine serum albumin, 0.1% sodium azide NaN ₃ , pH 7, 0.

2626S3 gar, hydroxypropylcellulose and similar high molecular weight materials.

Although in many cases it is sufficient to determine whether or not a candidate antigen is included in a substrate solution, it is most often desirable to perform a semi-quantitative or quantitative assay. If the immunocomplex is not itself colored, it appears necessary to label the antibody in some detectable manner.

One of the oldest methods of this kind is to label with radioactive isotopes such as iodine (1.25) or tritium and to determine the amount of radioactivity of the region containing the antibody-antigen complex by a radioactivity detector. Due to the fact that the handling of radioactive substances again requires a special laboratory, it is now preferable to label with fluorescent reagents, so that the antibody or the complex may be determined and measured on the basis of its fluorescence.

In particular, for proteins that are only present in low concentrations, the antibody is preferably coupled with a suitable enzyme and the reaction of a suitable substrate is utilized to catalyze the enzyme for the detection reaction. The evaluation can, as described above, be carried out either directly on the carrier material or after separation of the zone containing the complex and suspension in a suitable solvent, photometrically in the rock.

In addition, a semi-quantitative assay is achieved by admixing a defined amount of a specific enzyme-labeled and / or complex-specific adsorption agent in the carrier-fixed form to the end region of the carrier material and judging from the width of the absorption zone that develops .

In the following examples, rapid tests are explained in more detail, without being limited to the examples.

Example 1

Detection of hCG (human Chorionic Gonadotropin) in urine using test capillary.

Solutions

AND.

mg of indolylgalactoside is dissolved in 1 ml of methanol.

B.

Fab-anti-hCG / 3-galactosidase conjugate was made from sheep anti-hCG-serum [immunization: D. M. Weir, Handbook of Experimental Immunology, A 2.8; Fah digestion: M. E. Davis, A. J. Barret, R. M. Hernbry, J. of Immunological Methods, 21, 305 (1978)];

Conjugation: T. Kitagawa in Enzyme Immunol Essay, Editors: E. Ishikawa, T. Kawai, K. Miyai, Igaku-Shoiu, Tokyo-New York 1981) and to

Material:

[porous glass: glyceryl glass, Controlled Pore (Sigma, St. Louis)], 259 A pore diameter,

Glass diameter of 1 pulp particle size: 200 to 400 msh. capillary: length 12 cm, inner mm, closed on one side (up to 2 mm).

Capillary adjustment

The capillary is sealed at the lower end with some pulp and filled with porous glass (filling height 7 cm). 500 mg of the porous glass is mixed with 1 ml of solution A and then dried at 60. The porous glass capillary is now overlaid with a mixture of porous glass and indolylgalactoside substrate (filling height 1 cm). The pulp at the lower end of the capillary is impregnated with 3 μΐ of solution V and dried.

Test performance

The capillary is placed in the test urine. Within about 5 minutes, the liquid reaches the top layer of the substrate. The immunocomplex galactosidase reaches this substrate layer only in the presence of hCG. The colorless indolylgalactoside is hydrolyzed and in the presence of oxygen which can be visually.

let it react to indigo, recognizable by its blue color

Detection of hCG in urine using test strips.

Solutions:

AND.

ml 0.3% ogar-agar in 150 ml mM NaCl NaCl mM KP _h pH 7.2

4- 400 μϊ triton X — 100

4- 2.3 ml water

2 g of a 50% dispersion of polyvinyl propionate in water (Propiofan 70 D, BASF, Ludwigshafen)

B.

Sheep conjugate

Fab-Anti-hCG galactosidase from antiserum as in Example 1.

Materials (Daltosil, SP 300, pore size

Silica gel about 300 A, particle size 120-200 mesh, fa, Serva).

A mixture of silica gel and indolylgalactoside / a solution of 3 mg indolylgalactoside in 1 ml of methanol is charged with 250 mg of the above silica gel and the resulting slurry is dried in an air stream at 60-70 ° C.

Polycarbonate foil (Pokalon, Lonza, Lorrach).

Application

The following mixtures are produced:

(a) 8 ml of solution A - | - 1.5 g of silica gel

b) 1 ml of solution A 4- 0.19 g of a mixture of silica gel and indolylgalactoside

(c) 0,15 ml of solution A - 0,05 ml of solution 4 - 0,04 g of silica gel.

Using a spatula divided by partitions (rounded on one side), the mixtures and up to c in the contacting layers are applied to a 15 cm wide polycarbonate film (layer thickness of 400 ₍ m)).

Layer a 1 tin, layer c 0.5 cm, layer a 8 cm, layer b 1 cm and overlapping polycarbonate foil for gripping 4.5 cm.

The film is dried at 45 ° C and cut perpendicular to the application direction in 0.5 cm wide test strips (6 cm long).

Test performance

Description

Within 0 minutes, the liquid reached the uppermost end of the test strip and the presence of hCG leads to a color reaction as in Example 7.

Example 3

Influence of wetting agents on the properties of glass pores

Untreated porous glasses exhibit poor separation properties. Pretreatment brings considerable improvement. The glasses are preincubated with a solution consisting of 0.025% Tweeii 20, 10 mM sodium hydrogen phosphate Na-iHPO 3, 25 mM sodium chloride NaCl, 0.01% sodium azide NaNn, 30 min at pH 7.2, suctioned with suction and Dry in a drying cabinet at 35 ^° C until spray-proof.

As a measure of separation quality, the enclosed volume is determined by means of a low molecular weight substance, chlorphenol red, in filled 11 cm long capillary columns.

Porous glasses: Sigma, 120 to 200 mesh Filling: 4 μΐ of color solution, chromatography in water.

Capillary column filling height: 11 cm.

The test strips are immersed in the urine with their lower end.

pore diameter untreated material impregnated material color maximum at peak width color maximum at peak width 240 A 8,0 cm 1,2 cm 5 cm 0.8 cm 350 A 10.0, cm 2,0 cm 5 cm 1,2 cm 500 A 7,5 cm 4,0 cm 4.8 cm 1,2 cm 700 A 7,2 cm 1.7 cm 4,9 cm 1,2 cm 1000 A 10,3 cm 1.3 cm 4.8 cm 0.8 cm

According to the manufacturer's data, theoretically, that is to say, for a material carefully suspended in a liquid and equilibrated in columns, in this example 4.7 centimeters should be achieved.

Example 4

A number of inert impurities on the properties of the glass-cell division

The quality of separation depends on the chromatography speed and the chromatography speed. Too fast chromatography results in a reduction in the separation performance. The rate of chromatography in capillary columns filled with dry porous glass may be reduced by the addition of hydrophobic particles. The following table shows the effect of hydrophobised silica gel admixture [Octyl-SP 500, 40 to 100 μΐη. fa Serva / per capillary column elution rate (diameter 1 mm, length 10 cm)] with porous glass (Controlled Por Glass, 240 A, 200-400 mesh, fa, Serva).

Table

§ (W / W) Octyl— —SP 500 chromatography time min. 0 3.4 3 7.8 5 12 7 16 10 19 Dec 13 20 May 14 21 17 24 20 May 52 22nd 58 25 175

Example 5 Porous glass CPG, fa. Servos, 200 to 400 nieh, column fill height: 10 cm, deposition.

Molecular weight division for 3 μ! protein solution (2 mg protein / pre-treated glasses / ml), peak width: 0.5 to 0.7 cm. Solvent: water.

pore diameter

Cytochrome C

MG - 12,000 fluorescein labeled Antibody (Antialbumin) MG - 150,000 Ferritin

MG = 450,000 dextran blue

MG about 6 million fluorescein-labeled antibody (antialbumin) chromatography at 1 mg albumin / ml PES maximum at

240 A 35C A 5,3 cm 5,4 cm 6,5 cm 5,6 cm 7,1 cm 6,2 cm front front

1) 2)

6.5 4- 8.1 cm

1) an antibody

2) an antibody-antigen complex

Claims (15)

SUBJECT OF THE INVENTION CLAIMS 1. Method for carrying out immunochemistry rapid tests in which antigens react with antibodies to form immunocomplexes, characterized in that the test substrate is deposited on the lower part of the apparatus and chromatographed by solvent elution with a layer and the possible iiminocomplex is detected in the upper part of the apparatus. 2. A method according to claim 1, wherein the bottom of the device is filled with a substrate containing the antigen and is introduced into a solvent, for example water or an aqueous buffer solution, which capillaryly rises upwardly, dividing the antibody and immunocomplex. 3. The method of claim 1, wherein the antiuene-containing substrate solution is an eluent in chromatography. 4. The method of claims 1 to 3, wherein the immunocomplex itself or with it is detected. coupled labeling enzyme after chromatographic separation. 5. Apparatus for carrying out the method of item 1, comprising a capillary active layer consisting of a dry, porous, non-swellable, granular material, wherein the antibodies, antigens and imnoconconlexes are soluble compounds and the material exhibits molecular sieve properties, wherein the pores of the molecular sieve The sieves are larger than the antibody but smaller than the drive complexes formed from the antigens and the antibody. 6. The apparatus of claim 5, wherein the capillary active layer comprises a capillary column. Device according to claim 1, characterized in that the capillary-active layer is deposited on an inert carrier, for example a plastic film or glass. Device according to one of Claims 5 to 7, characterized in that the antibody is impregnated at the bottom of the molecular sieve. Device according to one of Claims 5 to 7, characterized in that the antibody is impregnated on the porous layer upstream of the capillary layer. Device according to one of Claims 5 to 9, characterized in that the antibody is labeled with a radioactive substance, a fluorescent substance or a chromophore. Device according to one of Claims 5 to 9, characterized in that the antibody is labeled with an enzyme. 12. The apparatus of claim 11, wherein the top of the material is impregnated with a reagent system for the marking system. Apparatus according to one of Claims 5 to 12, characterized in that the molecular sieve material comprises an inert hydrophobic fine-grained material, for example talc, hydrophobized glass or silica gel. Apparatus according to one of Claims 5 to 13, characterized in that the molecular sieve consists of a hydrophilic material, for example silica gel or porous glass, and / or is impregnated with a wetting agent, for example cationic, anionic or nonionic, such as glycol ethers, alkylsulfates, phenol ethers With 2-hydroxyethylamine lauric acid. Apparatus according to one of Claims 5 to 14, characterized in that the molecular sieve material is additionally impregnated with buffers and / or salts and / or complexing agents or other active substances.