FI78786B - IMMUNOCHEMICAL SNABBEST. - 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

1 78786

This invention relates to immunochemical rapid assays using chromatographic aids and methods for the immunochemical determination of suitable proteins.

The importance of immunochemical tests for the determination of hormones, proteins, pharmaceuticals, etc., for example in blood, urine and faecal samples, but also in clinical chemistry, for example in food analysis or in the analysis of water samples, is constantly growing.

The advantage of these experiments is the high specificity and affinity of the immunoreagents, with which even small amounts of the substrate to be determined can be determined with certainty among the higher concentrations of other, relatively similar compounds. Particularly detrimental to the layman are the numerous processing steps which, in traditional experiments, are time-consuming and which can only be carried out using abundant laboratory equipment. Therefore, in view of the wider use of immunochemical assays, it is desirable to have specific low-threshold assays that can be used without complex accessories and, where possible, without additional pretreatment and post-treatment of substrates, with short results.

Therefore, the object of the present invention was to develop a rapid test which makes the performance of immunochemical assays simple and fast even for a lay person.

For example, the following reactions are known in immunochemical experiments: 2 78786 1. Competitive Binding Method In an unknown amount of antigen or hapten (hereinafter abbreviated as "antigen") to be determined in this method, a well-known amount of labeled antigen is added and then reacted with a competitive solid support. with a sub-amount of antibody The solid phase and the liquid phase are separated, and the amount of antigen to be determined is obtained indirectly by determining the amount of labeled antigen in the solid or liquid phase.

2. IEMA Principle The antigen to be determined herein is reacted with an excess of labeled antibody, and in a second step, the unregulated antibody is bound to the corresponding antigen attached to the carrier. Again, after phase separation, the amount of antigen is obtained indirectly by determining the labeled substances in the solid or liquid phase.

3. Sandwich Principle In this method, a divalent antigen is converted with a solid-phase-bound antibody on the one hand and a labeled antibody on the other, and again after separating the liquid and solid phases, the labeled antibody concentration is determined from one of the two phases to give the divalent antigen concentration indirectly.

Common to all of these methods is that one of the reactants is bound, or subsequently bound to the support, to allow separation between the complex and the excess reagent. In addition to the fact that these bindings to the carrier involve additional costs and, above all, can alter the immunochemical properties, the implementation of these methods requires, as has already been stated, a relatively large amount of effort. As a result, they are not suitable for simple and inexpensive rapid test methods.

3,78786 Therefore, the rapid experiments of the invention should not use antibodies or antigens attached to carriers, but antibodies that act directly on the immunization, possibly in their labeled form. According to the invention, an antibody is understood to mean complete antibodies, but also active fragments, such as Fab fragments. The antibodies can be used in both polyclonal and monoclonal forms. This is surprisingly possible because the immunocomplexes of antibodies and antigens can be chromatographically separated on the basis of their different molecular weights or the different size of their unmodified antibodies or antigens, after which the corresponding determination of the chromatographically separated fractions is easily possible.

Gel filtration methods, for example using Sephadex: Sephacryl and Ultrogel columns, to separate proteins based on their different sizes are well known.

However, these methods are particularly demanding, so only trained personnel can use them with equivalent laboratory equipment. For example, the gel material must be swollen for several days in advance, the chromatography columns must be carefully filled, equilibrated and kept constantly moist with solvent to achieve good separation. In addition to the column, solvent storage vessels, pumps, fraction collectors, detector systems, etc. are still needed, and in addition, the time required to prepare the samples and perform the chromatography is several hours, so these methods are not suitable for rapid research.

It has now surprisingly been found that a capillary active layer of a dry, porous, swellable, granular material having molecular sieve-like properties (e.g., porous glass, kieselgel, and the like) can be used to rapidly separate substrates of similar molecular size when capillary the fluid transport caused by it is exploited. Depending on the pore diameters, the substrates chromatograph at a molecular size of 4,78786, corresponding to a slower or faster rate, with high molecular weight substances passing through the chromatography column immediately after the solvent front, because these substances are unable to penetrate the pores due to their molecular size. Thus, without prior cumbersome operations, i.e. without balancing the column in advance or without additionally using a pump to move the liquid, corresponding separations can be made in a relatively short time - 5-30 minutes with a column length of 5-20 cm. To avoid misbehavior of the column, the diameter of the column should be 0.5-5 mm, preferably 1-2 mm. Since immunocomplexes are generally substantially larger than the antibodies and antigens used to form them, using suitable molecular sieves, the pore diameter can be selected so that the immunocomplexes do not penetrate at all or penetrate only to a small extent, i.e., with little or no solvent front chromatography. as the antibodies are able to penetrate the pores, thereby retaining them, i.e. chromatographing substantially more slowly. Therefore, the above-mentioned dry capillary columns can be used for a rapid immunological test.

In this case, according to the invention, two different procedures are possible: 1. An excess of antibody can be added to the antigen-containing substrate and incubated beforehand. After successful conversion, the mixture is poured onto the column formed by the molecular sieve described above, after which the chromatographic separation is carried out either as a suitable solvent, i.e. usually with an aqueous buffer solution or with an excess of the reaction solution. Thereafter, the concentration of the immunocomplex in the solvent-free zone free of unmodified antibodies can be determined by any method known per se.

5,78786 2. It is even simpler for the user to use prefabricated columns, the lower part of which contains a molecular sieve material impregnated with a suitable antibody, and the upper part of which contains an unsaturated chromatographic material. When an antigen-containing substrate is now implanted in the antibody-saturated portion, an immunocomplex is formed in the mobile phase which, due to its size, cannot penetrate the pores of the molecular sieve, so it moves faster than the unmodified antibody retained in the pores of the stationary phase.

It seems very surprising that such a quick and useful separation is possible by such simple means, as the literature data would expect the liquid passing through the column to wet the dry column only incompletely, i.e. to form smaller and larger air sacs inside and between the particles. which would make separation impossible. On the other hand, it was expected that, especially in Method 2, the presence of diffusion slows down the formation of large immunocomplexes, which can only occur outside the pores, so that during a short contact lasting a few seconds, a sufficient reaction rate is not achieved.

The invention is described in more detail in the claims.

In a preferred embodiment of the invention, the other end of a glass or plastic capillary having an inner diameter of 0.5-5 mm, preferably 1-2 mm, and a length of 5-20 cm, preferably 10-20 cm, is closed with an absorbent, loose material. A layer of 0.2-10 mm thick molecular sieve material impregnated with the labeled antibody is placed on top of this barrier material, and the column is further filled with an unsaturated molecular sieve, and its upper end is again sealed with a permeable barrier material. When a labeled antibody cannot be detected directly, for example, by its color, fluorescence, or radioactivity, the top of the column may further comprise a detection zone containing, for example, corresponding reagents to produce the antibody used to label the antibody, e.g. detectable product.

If a few test solution is implanted at the lower end of such a column, after which the column is placed in a suitable developing solution, then the liquid travels upwards due to capillary forces, completely filling the contents of the column. In the presence of antigens, complex formation and separation of these complexes occurs at the upper end of the capillary column, as shown. Because the chromatography ends automatically after the column is completely wetted, special monitoring by the user is unnecessary. Especially in the case of a qualitative experiment, the result can be read even after a long time, and the solvent can even possibly be evaporated, and the column kept for documentation purposes.

In yet another preferred embodiment, the molecular sieve material is applied in a layer about 0.05-1 mm thick, optionally mixed with a binder, to an non-absorbent substrate, and this is divided into strips 5-10 mm wide and 50-150 mm long. Depending on the application, one end of these strips is further impregnated with corresponding antibodies, this end being used as a sample implanting head and the other end further impregnated with a reagent for detecting antibodies, which end is used to detect the formed antibody / antigen complexes. If the three zones are applied separately, care must be taken to ensure adequate capillary contact between them. However, it is also possible to first apply the layer-forming mass suspended in a suitable solvent, for example with a sweeping scraper, and allow it to dry, after which the layer is impregnated with ragens. Plastic sheets, such as polycarbonate, polyester, polyamide or polyethylene sheets, are preferably used as the impermeable substrate, however, a glass substrate may also be preferred.

As indicated above, the molecular sieves used for chromatography should be of non-swellable material. For this reason, it is preferred to use inorganic carriers, and in particular commercially available, different types of porous glasses and kieselgels with a certain pore size. However, the inner and outer surfaces of the inorganic carrier material may be coated with a suitable organic substance, for example a polyol, to make the adsorption properties of these carriers similar to those of an agar or cellulose gel, and to prevent negative effects on inorganic carrier antibodies or antigens. Since mainly aqueous solutions are suitable as solvents in chromatography, the materials used must be sufficiently hydrophilic.

If the preparation of the materials does not already guarantee this property, then the suspension of the carrier materials in an aqueous solution containing a wetting agent and a buffer before the actual use and, in turn, the drying of the granules thus impregnated has proved advantageous.

As the wetting agent, all known anionic, cationic or nonionic wetting agents can be used. The recommended concentrations used are 0.01-0.1%, lower concentrations can also be used when using naturally hydrophilic materials, higher concentrations can also be used as long as they do not affect the immunocomplex. Examples of wetting agents are glycol ethers, such as polyoxyethylene stearate or laurate, alkyl sulphates, such as dodecyl sulphate, fatty alcohol polyglycol ether, phenol ethers, for example 10,5-ethoxynonylphenol, cetyltrimethylammonium bromide and laurylethynamino bromide.

The other components of the impregnation solution roughly correspond to the components in the development solution to be used later, i.e. buffers, salts, complexing agents, sterilants, etc., which, as smaller molecular size components, chromatograph more slowly than the solvent front, thereby also achieving solvent separation.

It has further proved advantageous to mix the finest possible, inert and possibly hydrophobic packing mass with the chromatographic material, in particular talc, hydrophobicized glass, kieselgel and the like. This material on the one hand reduces the void space between the carrier particles, and thus leads to a better separation, and on the other hand slows down the velocity of the liquid phase, improving equilibrium as the diffusion between the mobile and stationary phases improves, which in turn improves the separation. In addition, the column filling is more homogeneous, which also avoids the possible formation of air sacs.

When selecting binders for the production of layered carriers, care must be taken to ensure that they lead to stable layers, but that they do not substantially alter the transport properties of the carrier material, i.e. in particular they must not clog pores. Surprisingly, this is achieved by using a mixture of a plastic dispersion in water, for example a dispersion of polyvinyl propionate or polyacrylate, and a highly water-soluble or swellable binder. Agar, hydroxypropylcellulose and other similar high molecular weight materials have been used successfully.

Although in many cases it is sufficient to determine whether or not the antigen in question is present in the substrate solution, in many cases it is desirable to perform a semi-quantitative or quantitative assay. If the immunocomplex is not in itself colored, then it is necessary to label the antibody in some demonstrable manner.

9,78786

One of the oldest such methods is labeling with radioactive isotopes, for example iodine (125) or tritium, and quantifying the radioactivity of the zone containing the antibody / antigen complex with a radioactivity counter. Because the handling of radioactive materials, in turn, requires special laboratories, labeling today is preferably performed with fluorescent dyes, allowing the antibody or immunocomplex to be determined and detected by fluorescence.

In the case of low-concentration proteins in particular, the antibody is preferably combined with a suitable enzyme, and the detection reaction uses the conversion of suitable substrates by the catalytic action of this enzyme. This detection reaction can be performed either directly in the support material as described above, or it can be performed photometrically in the cuvette after separation of the complex-containing zone and suspension in a suitable solvent.

In addition, semi-quantitative determination is possible by mixing a certain amount of a specific adsorbent for the labeling enzyme and / or the carrier-adherent complex in the final zone of the carrier material, whereby the initial amount of enzyme in the immunocomplex can be deduced from the width of the adsorption zone.

The rapid experiments according to the invention are illustrated in more detail in the following examples, without, however, limiting the invention.

examples

Example 1 Detection of hCG (human Chorionic Gonadotropin) in urine by experimental capillary_

Solutions A. Dissolve 3 mg of indolylgalactoside in 1 ml of methanol.

B. Fab-Anti-hCG-β-galactosidase conjugate was prepared from sheep anti-hCG serum (Immunization: DM Weir, Handbook 10 78786 of Experimental Immunology, A 2.8; Fab cleavage: ME Davis, AJ Barrett, RM Hernbry, J. of Immunological Methods, 2, 305 (1978); Conjugation: T. Kitagawa in Immuno Assay, Edited by E. Ishikawa, T. Kawai, K. Miyai, Igaku-Shoiu, Tokyo - New York 1981), and was dissolved in 575 U / ml in a solution of 10 mM KP 2, 5 mM MgCl 2, 25 mM NaCl, 2% sucrose, 0.5% bovine blood albumin, 0.1%: from NaN3, pH 7.0.

Materials ^ / Porous glass: Glyceryl-Glas, Controlled Pore (Sigma, St. Louis2? R pore diameter 259 Å, particle size 200-400 me sh.

Glass capillary: length 12 cm, inner diameter 1 mm, closed on one side with cellulose wadding (height 2 mm).

Filling the capillary

The capillary is sealed at its lower end with a small amount of cellulose wadding and filled with porous glass (filling height 7 cm). 500 mg of porous glass are mixed with 1 ml of solution A and then dried at 60 ° C. A new layer is formed in this capillary partially filled with porous glass with this mixture of porous glass and indolylgalactoside substrate (filling height 1 cm). The cellulose wad at the lower end of the capillary is saturated with 3 yU of solution B and dried.

Performing the test

The capillary is placed in the sample urine. Within about five minutes, the liquid reaches the upper substrate layer. Only in the presence of hCG does galactosidase from the immunocomplex reach this substrate layer. The colorless indolyl galactoside hydrolyzes and, in the presence of oxygen, turns into indigo, which becomes visually detectable blue due to its coloration.

li 78 786

Example 2 Detection of hCG in urine by test strips Solutions A. 20 ml of 0.3% Agar in 150 mM NaCl 10 mM KPif pH 7.2 + 400 Triton X-100 + 2.3 ml water + 2 g 50% aqueous polyvinylpropionate dispersion (Propiofan 70 D, BASF, Ludwigshafen) B. Fab-Anti-hCG-β-galactosidase conjugate derived from sheep anti-serum as in Example 1.

materials

Kieselgel (Daltosil, SP 300, pore size about 300 A, particle size 120-200 mesh, manufacturer: Serva).

A mixture of silica gel and indolyl galactoside (a solution prepared by placing 3 mg of indolyl galactoside in 1 ml of methanol is mixed with 250 mg of the above-mentioned silica gel, and the paste thus obtained is dried with a stream of air at 60-70 ° C).

Polycarbonate film (Pokalon, manufactured by Lonza, Lörrach). The deposition

Prepare the following mixtures: (a) 8 ml of solution A + 1,5 g of silica gel (b) 1 ml of solution A + 0,19 g of the mixture between the gel gel and indolylgalactoside (c) 0,15 ml of solution A + 0,05 ml of solution B + 0, 04 g Kiesel gel.

Mixtures a-c are applied to the polycarbonate film with a width of 15 cm (layer thickness 400 μm) by means of a split scraper with partition walls (one side rounded). In this case, the widths of the layers and their order are as follows: 12 78786

Layer a 1 cm, layer c 0.5 cm, layer a 8 cm, layer b 1 cm and the width of the remaining polycarbonate film acting as a contact surface is 4.5 cm.

The film is dried at 45 ° C and cut perpendicular to the layers into test strips 0.5 cm wide (length 6 cm).

Performing the test

The lower end of the test strip is dipped in urine.

Description

Within six minutes, the liquid has reached the top of the test strip, and the presence of hCG results in a color reaction, as in Example 1.

Example 3

Effect of wetting agents on the resolving power of porous glass The separating properties of porous, untreated glass are poor. Pretreatment provides significant improvement. The glasses are pretreated for 30 minutes in a solution of 0.025% Tween 20, 10 mM Na 2 HPO 2, 25 mM NaCl, 0.01% NaN 2, pH 7.2, after which they are dried on a suction filter and dried in an oven at 35 °. collapse to dryness.

To measure the goodness of the separation, the elution volume is determined with a small molecular weight substance, chlorotenol red, on a 11 cm long, filled capillary column.

Porous glass: About Sigma, 120-200 mesh.

Batch: 4 μl of dye solution, chromatography was performed with water. Capillary column filling height: 11 cm.

13 78786 Untreated Impregnated material material

Peak color maximum peak color maximum peak diameter point width point 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 700A 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 information provided by the manufacturer, in this example the value of 4.7 cm should theoretically be reached, i.e. with the material carefully dissolved in the liquid and equilibrated in the column.

Example 4

Effect of inert substances on the separation properties of porous glasses_

The good separation of gel chromatography also depends on the speed of the chromatography. Too fast chromatography results in reduced separation. The speed of chromatography on a dry, porous glass-filled capillary column can be reduced by adding hydrophobicized particles to the filler. The following table shows the effect of mixing according to a hydrophobicized silica gel (Octyl-SP 500, 40-100 μm, from Serva) on a capillary column (diameter 1 mm) filled with porous glass (Controlled Pore Glass, 240 A, 200-400 mesh, from Serva). , length 10 cm) to the elution rate.

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Table% (W / W) Required for chromatography

Octyl-SP 500 time, min_ 0 3.4 3 7.8 5 12 7 16 10 19 13 20 14 21 17 24 20 52 22 58 25 175

Example 5

Separation on the basis of molecular weight with pretreated glass Porous glass CPG, from Serva, 200-400 mesh, column loading height: 10 cm, batch: 3 μl of protein solution (2 mg protein / ml) each, peak width: 0.5-0.7 cm. Solvent: water.

Pore Maximum Point

diameter_240 A_350 A

Cytochrome C

MP = 12000 5.3 cm 5.4 cm

Fluorescein-labeled antibody (anti-albumin) MP = 150,000 6.5 cm 5.6 cm

Ferritin MP = 450,000 7.1 cm 6.2 cm

Dextran blue MP about 6 mill. English Translation:

Fluorescein-labeled antibody (anti-albumin) in chromatography 1 mg 1) 2) albumin / ml PBS 6.5 + 8.1 cm 1) antibody, 2) antibody / antigen complex

Claims (14)

A product for making an immunochemical assay, characterized in that it is composed of a capillary-active coating of a dry, porous, non-curing granular material having molecular properties, the molecular weight of which permeates pore penetration into the pores of the soluble antibodies used. immunochemical pathway of antigens, but does not allow or even to a small extent permeate the pores of soluble immunocomplexes formed by the antigen and the body. Product according to claim 1, characterized in that the capillary active layer comprises a capillary column with a diameter of 0.5-5 mm and a length of 5-20 cm. Product according to claim 1, characterized in that the capillary active layer is on an inert carrier material and is 0.05-0.5 mm thick, 5-10 mm wide and 5-20 cm long. Product according to any one of claims 1-3, characterized in that the lower molecular weight of the molecular sieves is impregnated with antibodies, or antibodies are a porous acrylic used in advance. Product according to any of claims 1-4, characterized in that the antibody is labeled radioactive, fluorescent or labeled with a color-forming substance. Product according to any one of claims 1-4, characterized in that the antibody is labeled with an enzyme. 7. Product according to claim 6, characterized in that the upper part of the material is impregnated with a reagent atom with which the enzyme used for marking reacts to detect the reaction.