DD234495A5 - IMMUNECHEMICAL QUICK TEST - 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

AP G Ol N / 269 839 4 64 632/11

Device for carrying out immunochemical rapid tests Field of application of the invention

The present invention relates to a device for carrying out rapid immunochemical tests using chromatographic aids and methods for the immunochemical determination of suitable proteins.

Characteristic of the known technical solutions

Immunochemical tests are becoming increasingly important for the determination of hormones, proteins, pharmaceuticals, etc., for example in blood, urine and stool samples, but also outside of clinical chemistry, for example in food analysis or in the analysis of water samples.

The advantage of these tests lies in the high specificity and affinity of the immune reagents, which makes it possible to safely determine even small amounts of the substrates to be detected in addition to higher concentrations of relatively similar other compounds. A disadvantage, especially for non-professionals, are the many handling steps that take a long time in conventional testing and can usually only be performed with extensive laboratory equipment. For, c ine wider application of immunochemical provisions, it is .wünschenswert to have specific tests with low detection limit for disposal that the without complicated additional equipment and, if possible without additional pre- or post

Substrates are used and display the result in a short time.

The object of the present invention was therefore to develop rapid tests with which immunochemical determinations can be carried out easily and quickly, even for the layman.

For immunochemical tests, for example, the following reaction modes are known:

1. Competitive binding procedure

In this method, to an unknown amount of an antigen or hapten to be detected (im

hereinafter referred to as "antigen") is added to an accurately known amount of an appropriately labeled antigen which is subsequently reacted in competition with a deficiency of a corresponding antibody fixed to a solid support. Solid phase and liquid phase are separated and deduced by determining the amount of labeled antigen in the solid or liquid phase on the amount of the antigen to be determined

2. IEMA principle

Here, the antigen to be determined is reacted with a labeled antibody in excess, and in a second step the unreacted antibody is bound to a corresponding carrier-fixed antigen. Again, after separation of the phases by determination of the label in the solid or liquid phase, the amount of antigen can be deduced.

3. sandwich principle

In this method, a bivalent antigen is reacted on the one hand with an antibody bound to a solid phase and on the other hand with a labeled antibody and again after separation of the liquid and solid phase in one of the two phases determines the concentration of the labeled antibody and thus the concentration of the bivalent Antigen closed.

All of these methods have in common that one of the reactants is carrier bound or subsequently bound to allow separation between complex and excess reagent. Apart from the fact that this carrier fixation causes additional costs and above all can change the immunochemical properties, these methods, as already stated above, require a relatively high expenditure in their implementation. They are therefore not suitable for simple, cheap Schenlltestverfahren.

Furthermore, methods for the separation of proteins due to their different sizes are well known as gel filtration on, for example, Sphadex, Sephacryl, and Tytolysis columns. However, these methods are characterized by special requirements, so that they can be performed only by semi-skilled persons with appropriate laboratory equipment. The gel material MUS> for example, several days will be pre-swelled, the chromatographic columns must be carefully filled equilibrated with the solvent and constantly feuc ^nt be maintained in order to achieve a good separation. In addition, in addition to the column, reservoirs for the solvents, pumps, fraction collectors, detector systems, etc. are required and, in addition, sample

preparatory and chromatographic times of several hours so that these methods are not suitable for a rapid test study.

Object of the invention

The aim of the invention is to provide new methods and suitable devices for carrying out the methods with which immunochemical tests for the determination of hormones, proteins, pharmaceuticals, etc. can be carried out in a simple manner within a short time, even by semi-skilled assistants.

Presentation of the essence the invention

The invention is based on the object for the rapid tests no carrier-fixed antibodies or antigens, but to use the antibodies directly obtained in the immunization optionally in their labeled form. Antibodies according to the invention are to be understood as meaning the complete antibodies but also active fragments, for example Fab fragments. Antibodies can be used in both polyclonal and monoclonal form. Surprisingly, this is possible because the immune complexes of antibody and antigen due to their different molecular weight or their different size of unreacted Antikö.vern, or antigens can be separated by chromatographic means and a corresponding detection intray chroraatographisch separated fractions then easily possible »

Surprisingly, it has now been found that a capillary-active layer, which consists of a dry, porous, non-swelling, granular material with Molekularsiebeigenschaften (z, B * pore glass, silica gel o. Ä.) Can use for a rapid separation of substrates of appropriate molecular size Depending on the pore diameter, the substrates chromatograph more or less rapidly according to their molecular size, whereby high molecular weight substances, which can not penetrate into the pores due to their size, are chromatographed directly behind the solvent front without previous great effort, d. H. without first equilibrating these columns

or to move the liquids additionally with pumps to perform within relatively short times - 5 to 30 minutes for column lengths of 5 to 20 cm - appropriate separations. In order to avoid "skewing" of the column, the diameter of the column should be 0.5-5 mm, preferably 1-2 mm. Immune complexes are generally much larger than theirs. When using suitable antibodies and antigens, it is possible, when using suitable molecular sieves, to choose the pore diameter such that the immune complexes do not or only slightly penetrate the pores, i. chromatograph with the solvent front or just behind it while the antibodies can penetrate into the pores and thus be retained or chromatograph much slower. The aforementioned dry capillary columns can therefore be used for a rapid immunological test.

According to the invention, two different procedures are thus possible:

' 1. . The antigen-containing substrate may be spiked with an excess of the antibody and preincubated. After the reaction is complete, the mixture is then applied to a molecular sieve column described above and either with a suitable solvent, i.

normally chromatographed with an aqueous buffer solution or with an excess of the reaction solution. In. the concentration of the immune complex can then be determined by one of the methods known per se, the solvent front free of the unreacted ^L antibody.

2. It is even easier for the user to use prefabricated columns containing in their lower part molecular sieve material impregnated with suitable antibodies and containing in the upper part unimpregnated material for chromatography.

If the part impregnated with the antibody is now loaded with antigen-containing substrate, the immune complex is formed in the mobile phase, which due to its size can not penetrate into the molecular sieve pores and thus migrates faster than the unreacted antibody which is in the stationary phase in the Pores remains.

It seems extremely surprising that with such simple means a rapid and useful separation is possible because, according to the literature, it had to be expected that the dry column would be imperfect due to the passing liquid. would be wetted to form more or less large air entrances within and between the particles, which would make separation impossible. On the other hand, it was believed that especially in Method 2, the formation of large immune complexes, which can only take place outside the pores, is slowed down by diffusion processes, so that a sufficient reaction rate does not occur within the short contact time of a few seconds.

The invention is characterized in the claims.

In a preferred embodiment of the invention, a capillary made of glass or plastic with a r.

Inner diameter of 0.5 to 5 mm, preferably 1 to 2 mm and a length of 5 to 20 ctn, preferably 10 to 20 cm closed at one end with an absorbent, loose material. A 0.2 to 10 mm thick layer of the molecular sorbent material impregnated with a labeled antibody is placed on this occlusion material and the column is filled with further non-impregnated molecular sieve and sealed at the upper end with a permeable sealing material. »If the labeled antibody can not be detected directly , For example, due to its color, fluorescence or radioactivity, the upper part of the column may additionally contain a detection zone in which, for example, appropriate reagents are included to produce a detectable product with the label of the antibody, such as a coupled enzyme.

In a particularly advantageous embodiment of the invention, the molecular sieve material is additionally admixed with an inert, hydrophobic, fine-grained material.

In another embodiment of the device according to the invention, the molecular sieve material is hydrophilic and / or impregnated with a wetting agent in an amount of 0.01 to 0.1%.

It is also advantageous if the molecular sieve material is additionally impregnated with buffers and / or salts and / or Koraplexbildnern or other active ingredients.

If such a column is sprayed on at the lower end with a few μl of the solution to be investigated and then placed in a suitable developing liquid, then the liquid migrates up to complete covering of the contents of the column due to the capillary forces. at

-7a

Presence of the antigen occurs, as described, for complexation and separation of this complex at the top of the capillary column. Since the chromatography with the complete humidification is finished automatically, a special monitoring by the user is not necessary. The result can be read in particular in a qualitative test even after a long time and possibly even evaporate the solvent and the column are kept for documentation purposes.

\ In a further preferred embodiment which is

Molecular sieve material in a -0.05 to 1 mm thick layer optionally spread with the addition of a binder on a non-absorbent carrier and this divided into 5 to 10 mm wide, 50 to 150 mm long strips. Depending on the intended use, one end of these strips is additionally impregnated with appropriate antibodies and serves to apply the sample, and the other end is additionally impregnated with detection reagents for the labeling of the antibody and used to detect the antibody / antigen complexes formed. If the three zones are applied separately, care must be taken that there is sufficient capillary contact between them. However, it is also possible, first, the coating composition, in a suitable solvent

^ 'is suspended, for example with a. Spread and dry the squeegee and then re-impregnate the reagents. As an impermeable support preferably strips of plastics, such as polycarbonate, polyester, polyamide or polyethylene are used, but glass support can also be advantageous.

As already stated above, the molecular sieves used for the chromatography should consist of non-swelling material. For this reason, preference is given to inorganic carriers and, in particular, the various commercially available types of pore glass or silica gels having a defined pore size. However, the inner and outer surfaces of the inorganic support material may be coated with a suitable organic substance, for example

be coated with a polyol which gives the adsorption properties of an agar or cellulose gel and prevents negative effects of the inorganic carrier on the antibodies or antigens. As solvents for chromatography are predominantly aqueous solutions, the materials used must be sufficiently hydrophilic. If this is not already ensured by the preparation of the materials, it has proven to be advantageous to suspend the support materials before processing in an aqueous solution containing wetting agents and suitable buffers and to dry the thus impregnated granules again.

As wetting agents, it is possible to use all known anionic, cationic or nonionic wetting agents. Concentrations of 0.01 to 0.1 % are preferably employed, lower concentrations are also useful, in particular, for naturally hydrophilic materials, higher concentrations may also be used as long as they do not affect the immune complexes. As a network center! Examples are glycol ethers such as polyoxyethylene stearate or laurate, alkyl sulfates such as Dodezylsulfat, Fettalkoholpolyglykoläther, .Phenoläther, for example, 10,5-ethoxy-nonylphenol, Cetyltrimethylammoniumbromid and

Lauric acid bis-2-hydroxyethylamiη called.

The remaining constituents of the impregnating solution correspond approximately to the constituents contained in the later developing solution, i. Buffers, salts, complexing agents,

-10 -

Sterilization agents, etc., which are slower to chromatograph as low molecular weight constituents than the solvent front, whereby it is achieved that also prevail at the solvent front stationary conditions. '5. ,

Furthermore, it has proved to be advantageous to the chromatographic material: in addition as finely powdered, inert and possibly hydrophobic filling compound to mix, with talc, hydrophobized glass, silica gel and the like have proven particularly useful. This material reduces on the one hand the exclusion volume between the carrier particles and thus leads to a better separation and slows down the other hand, the running speed of the liquid phase, whereby the equilibration by improving the diffusion between the mobile and stationary phase and thus in turn improves the separation efficiency. In addition, the filling of the column is more homogeneous, which additionally avoids any occurrence of air bubbles.

When selecting the binders for the production of coated carriers, care must be taken that they on the one hand lead to a stable coating, but on the other hand must not significantly alter the running properties of the carrier material, i. especially do not clog the pores. Surprisingly, this is achieved by using an aqueous plastic dispersion, for example polyvinyl propionate or polyacrylate, mixed with a readily water-soluble or swellable binder. Agar-agar, hydroxypropylcellulose and similar high molecular weight materials have been used successfully.

- 11 -

Although it is sufficient in many cases to determine whether the antigen in question is contained in the substrate solution or not, it is desirable in most cases, to perform a semi-quantitative or quantitative determination. Unless the immune complex is not inherently colored, it therefore proves necessary to label the antibody in any detectable manner.

One of the oldest methods of this kind is the labeling with radioactive isotopes, for example with iodine (125) or tritium and determination of the amount of radioactivity of the zone containing the antibody / antigen complex by means of a radioactivity scanner. Since the handling of radioactive substances again requires special laboratories, it is nowadays preferable to effect the labeling with a fluorescent dye, so that the antibody or the immune complex can be determined and measured by its fluorescence.

In particular, for proteins which are present only in low concentration, the antibody is preferably coupled with a suitable enzyme and the reaction of suitable substrates under the catalytic action of this enzyme used for the detection reaction. This evaluation can, as already described above, be carried out either directly on the support material or after separation of the zone containing the complex and suspension in a suitable solvent by photometry in a cuvette.

In addition, a semiquantitative determination is possible by adding to the end zone of the support material a defined amount of a specific adsorbent for the labeling enzyme and / or the complex in

-12-

carrier-fixed form and inferred from the width of the developing absorption zone on the original amount of enzyme in the immune complex.

embodiment

The invention is explained in more detail below with reference to some examples. In the following examples, the quick tests according to the invention are explained in more detail, without any limitation being thereby intended,

Examples Example 1

Detection of hCG (human chorionic gonadotropin) in urine

by means of test capillary

solutions

A »3 mg of indolylgalactoside are dissolved in 1 ml of methanol.

B. Fab anti-hCG-β-galactosidase conjugate was prepared from sheep anti-hCG serum (Immunization: D.M.

Weir, Handbook of Experimental Immunology, A 2.8; Fab cleavage: M.E. Davis, A. ö · Barrett,

R. Μ · Hernbry, D. of. Immunological Methods, 2_1 305 (1973); Conjugation.; T. Kitagawa in Enzyme Immuno Essay, editors: E. Ishikawa, T. Kawai, K. Miyai, Igaku-Shoiu, Tokyo - Nwe York 1981) and 575 U / ml in 10 mM _± 5 mM MgCl ₂ , 25 mM Na 017 * 2 % sucrose, 0.5 % bovine serum balm, 0.1 % NaN ₃ , pH 7.0.

-13- materials

(Pore Glass: Glyceryl Glass, Controlled Pore, 259Å pore diameter, particle size: 200-400 mesh.

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

Equipping the capillary

The capillary is sealed with some pulp at the bottom and filled with pore glass (filling height 7 cm). 500 mg of pore glass are mixed with 1 ml of solution A and then dried at 60 C. The capillary equipped with pore glass is now covered with this pore glass / indolyl galactoside substrate mixture (filling level 1 cm). The pulp at the bottom of the capillary is soaked in 3 μl of solution B and dried.

Test procedure

The capillary is placed in the urine to be tested. Within approx. 5 minutes, the liquid reaches the upper substrate layer. Only in the presence of hCG does galactosidase from the immune complex reach this substrate layer. The colorless indolyl galactoside is hydrolyzed and converted to indigo in the presence of oxygen; the visuall becomes recognizable by blue coloring.

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Example 2

Detection of hCG in urine using test strip solutions

A. 20 ml 0.3 % agar agar in 150 mM NaCl, 19 mM ΚΡ _± , pH 7.2 + 400, Ul Triton X-100 + 2.3 ml water + 2 g of a 50% polyvinyl propionate dispersion in water (Propofan 70 D)

B. Fab anti-hCG-β-galactosidase conjugate from sheep anti-serum as in Example

materials

Silica gel, (Daltosil, SP 300, pore size approx. 300 A, particle size 120 to 200 mesh). Silica gel Indolylgalactosidgemisch (a solution of 3 mg in 1 ml of methanol indolyl galactoside wirdmit 250 mg of the above silica gel was added and the slurry obtained at 60 to 70 ⁰ C in flowing air dried).

Polycarbonate film (Pokalon).

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Coating The following mixtures are prepared:

a) 8 ml of solution A plus 1.5 g of silica gel

b) 1 ml of solution A plus 0.19 g of silica gel indolylgalactoside mixture

c) 0.15 ml of solution A plus 0.05 ml of solution B plus 0.04 g of silica gel

With a doctor blade divided by partitions (rounded on one side), the mixtures a to c in contacting layers are spread on a 15 cm wide polycarbonate film (layer thickness 400 μm). The layer widths and order are as follows:

Layer a 1 cm, layer c 0.5 cm, layer a 8 cm, layer b 1 cm and protruding polycarbonate foil as a handle 4.5 cm »

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

Testdurchfuhrung

The test strip is placed in the urine with the lower end.

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Within 6 minutes, the liquid has reached the uppermost end of the test strip and the presence of hCG leads to the color reaction as in Example 1,

Example 3

Influence of wetting agents on the separation properties of pore glasses

Pretreatment brings significant improvements "The glasses are preincubated with a solution of 0.025 % Tween 20, 10 mM Na ₂ PO ₄ , 25 mM NaCl, 0.01 % NaN ₃ , pH 7.2 for 30 minutes , Suctioned on a suction filter and dried in a drying oven at 35 C until free-flowing.

As a measure of the quality of the separation, the inclusion volume is determined with a low molecular weight substance, chlorophenol red, in 11 cm long filled capillary columns. Pore glasses: 120 to 200 mesh,

Application: 4.ul color solution, chromatography in water, filling height of the capillary column: 11 cm.

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untreated peak waterproof it material width material pore color maximum 1.2 cm Farbraaximum peak knife at 2.0 cm at width 240A 8.0 cm 4,0 cm 5 cm 0.8 cm 350 A 10.0 cm 1,7 cm 5 cm 1.2 cm 500 A 7.5 cm 1.3 cm 4.8 cm 1.2 cm 700 A 7.2 cm 4.9 cm 1.2 cm 1000 A 10.3 cm 4.8 cm 0.8 cm

According to the manufacturer one should · theoretically, d. H. in the case of material carefully suspended in liquid and equilibrated in columns, in this example reach 4.7 cm,

Example 4

Influence of inert admixtures on the separation properties of pore glasses

The separation quality in gel chromatography also depends on the speed of the chromatography. Too fast chromatography leads to reduced separation efficiency. The speed of the chromatography in a capillary column filled with dry pore glass can be reduced by admixing hydrophobized particles. The following table shows the influence of the incorporation of hydrophobized silica gel (Octyl-SP 500, 40 to 100 μm) on the running speed of a capillary column ( 1 mm diameter, 10 cm in length) with pore glass (Controlled Pore Glass, 240 A, 200 to 400 mesh).

-IS table

% (W / w) Example 5 chromatography time 3.4 Octyl-SP 500 Minute 7.8 O 12 3 16 5 19 7 20 10 21 13 24 14 52 17 58 20 125 22 25

Separation by molecular weight with pretreated glasses

CPG pore glass, 200 to 400 mesh, filling height of the columns: 10 cm, application: in each case 3, ul of a proton solution (2 mg protein / ml), peak width: 0.5 to 0.7 cm, solvent: water,

- l!

Pore diameter

Maximum at

A ^; ·

350 A

Cytochrome C MW = 12,000

5.3 cm

5,4 cm

Fluorescein-labeled antibody (anti-albumin)

MG = 150000 6.5 cm

¹⁰ ferritin

MG = 450000. 7.1 cm

5,6 cm

6, 2 cm

15

Dextran blue MG approx. 6 mill.

Fluorescein-labeled antibody (anti-albumin), chromatography in 1 mg albumin / ml PBS

Γ * \ 2 ο

front

front

D 2) 6.5 + 8.1 cm

1) antibody, 2) antibody / antigen complex

25 3o 35

Claims (14)

-20-invention-statement 1. A device for carrying out rapid immunochemical tests, characterized in that it consists of a capillary-active layer of a dry, porous, non-swelling, granular material having molecular sieve properties, the pore size of the molecular sieve permitting penetration of the soluble antibodies used for immunochemical detection of the antigens, but does not or only slightly penetrate the soluble imbrunk complexes formed from antigen and antibody, 2 «device according to item 1, characterized in that the capillary-active layer consists of a capillary column with a diameter of 0.5 to 5 mm and a length of 5 to 20 cm. 3, device according to item 1, characterized in that the capillary-active layer is on an inert support material and 0.05 to 0.5 ram thick, 5 to 10 mm wide and 5 to 20 cm long. 4, device according to one of the items 1 to 3, characterized in that the antibody is impregnated on the lower part of the molecular sieve, or is in an upstream porous layer, 5, Device according to one of the items 1 to 4, characterized in that the antibody is radioactive, fluorescent or labeled with a chromophore. -21- 6. Device according to one of the items 1 to 4, characterized in that the antibody is labeled with an enzyme, 7. Device according to item 6, characterized in that the upper part of the material is impregnated with a reagent system which enters into a detectable reaction with the marking enzyme. 8. Device according to one of the items 1 to 7, characterized in that the molecular sieve material is additionally admixed with an inert, hydrophobic, fine-grained material. 9. Device according to one of the items 1 to »8, characterized in that the molecular sieve material is hydrophilic and / or impregnated with a wetting agent in fine amount of 0.01 to 0.1 % . 10. Device according to one of the items 1 to 9, characterized in that the molecular sieve material is additionally impregnated with buffers and / or salts and / or complexing agents or other active ingredients. 11. A method for detecting an immunochemical reaction by complex formation of antigens with antibodies, characterized in that the mixture is chromatographed on a device according to item 1 to 3 with a suitable solvent and detects the optionally formed immune complex in the upper part of the device. -22- 12. The method according to item 11, characterized in that an apparatus according to item 4 is applied in the lower part with an antigen-containing substrate and placed in a suitable solvent which rises capillary through the device and chromatograpically separates antibodies and the immunocomplex optionally formed, 13. The method according to item 12, characterized in that the antigen-containing substrate solution also serves as eluent for the chromatography, 14. The method according to any one of items 11 to 13, characterized in that the immune complex itself or a labeling enzyme coupled thereto after the chromatographic separation is detected.