DE2733380A1 - Immunological assay using carrier bound reactant - incubated with second reactant and then with labelled reactant specific for the second - Google Patents

Abstract

Method comprises first contacting an inert, water-insoluble carrier having a flat surface coated with the first reaction partner (I) with a soln. of the second reactant (II). After a suitable time, the carrier is removed and contacted with a third, labelled reactant (III) which has immunological specificity for (II). The amt. of label bound is then detected or determined. Pref. carriers are 10-100 mu thick films of methyl methacrylate homo- or copolymers to which (I) is bonded covalently. Used esp. for quantitative analysis of body fluids; examples show analysis of anti-human immunoglobulin and rabbit anti-DNA. Simple and rapid process with very high specificity is provided.

Description

The invention relates to a method for qualitative and

especially for the quantitative determination of immunological reaction partners and a device for its implementation.

In immunological analysis processes with a carrier-bound reaction partner a sought antigen or antibody binds specifically immunologically a carrier-bound antibody or a carrier-bound antigen. There can the sought antigen or the sought antibody with a marker feature carrying antibodies specifically directed against the partner sought will. As a rule, the inner surfaces of test tubes or Used beads.

You can make of different materials. e.g. made of glass, plastic or cross-linked carbohydrates. Serve as a marking feature, for example Isotopes, enzymes or fluorescent substances. Enzymes and fluorescent substances have the advantage over isotopes that no radiation exposure occurs and that the measuring devices for the detection of an enzyme reaction or a fluorescent radiation require significantly less investment. With fluorescent substances as a marker is immediately after completion of the immunological reaction the measurement of the fluorescence intensity as a parameter for the amount of bound material and thus also as a parameter for the amount of the sought antigen or the sought Antibody possible. In the case of enzymes as a marker, the immunological Reaction to join chemical reactions for the detection of the enzyme. With help of fluorescent substances as a marker of reactants of a immunological reaction and with the help of corresponding reaction partners, which are bound to a carrier can thus be carried out quickly Detection method for a sought-after immunological reaction partner ("fluoroimmunoassay") build up.

In particular with regard to a sensitivity of Systems with carrier-bound reaction partners showed those with spherical ones Particle bound antigens less satisfactory. One has therefore tried flat carrier materials made of plastic with selected adsorption behavior for Determination of adsorbed reactants to be used. Since plastics with regard to the adsorption usually do not have a high specificity, this method is also flawed.

The task was therefore to create a simple, fast, highly specific To elaborate procedures for the immunological determination of reaction partners and To manufacture devices for performing immunological analysis.

It was found that carriers with a claner surface that are homogeneous are coated with the immunological reaction partner (antigen or antibody), the requirements of avoiding the disadvantages mentioned of the known methods are sufficient.

The invention accordingly relates to an immunological analysis method with a carrier-bound immunological reaction partner, characterized in that that a water-insoluble carrier indifferent to the reaction, which a flat surface coated with a first immunological reaction partner possesses, in contact with a solution containing a second reactant is brought, after a sufficient reaction time, the contact is canceled and the carrier carrying the reactants with a third, a marking agent carrying reaction partner is brought into contact, its immunological specificity is directed against the second reactant, after which the amount of bound Marking agent is detected or determined.

A plane or flat surface in the sense of the invention is a surface which corresponds to the focal plane in a given optical lens system.

The method shows particular advantages when using a fluorescent one Marking agent and a carrier which can be used for microscopic observation of the reaction result is suitable. Come as a carrier within the meaning of the invention therefore above all plane-parallel shaped bodies made of inorganic or organic material into consideration, preferably synthetic polymers, produced by casting, extrusion or Injection molding processes can be produced. Inorganic polymers such as glass; organic Homo- and copolymers of vinyl compounds such as olefins, vinyl acetate, vinyl chloride, Vinylidene chloride, tetrafluoroethylene, styrene, acrylates, methacrylates, formaldehyde and / or cyclic acetals and also polycondensates such as polyesters, polycarbonates or polyamides are used for the purposes of the invention.

With regard to their suitability in fluorescence test systems, preference is given to Homo- and copolymers are used that have no intrinsic fluorescence and therefore significantly increase the sensitivity of the analytical method. Particularly beneficial Homo- and copolymers of methyl methacrylate turn out to be.

The binding of the immunological partner to be fixed on the carrier can be done both adhesively and covalently.

The covalent bond is preferred, since this increases the stability of the carrier-bound reaction partner is much larger. As possible covalent Types of binding are those described in the literature, for example those made by H.D. Orth and W. Brümmer in Angew. Chem. 84 (1972), p. 319 - 330 and by J.H. Silman, E. Katchalski in Am. Rev. Biochem. 35 (1966), p. 873 ff, are listed.

For covalent binding of the protein to homo- and copolymers of The so-called azide method is preferably used for methyl methacrylate. As a carrier Injection-molded sheets or foils, in particular sequences 10-100 ß thick, are preferred. These moldings are reacted with hydrazine hydrate while maintaining their shape the acid hydrazide that forms is converted into the acid azide with sodium nitrite.

In an alkaline solution, a protein is then found that has the immunological Represents reactants at room temperature covalently to the surface of the support bound.

To create an adhesive bond, the carrier is put into the solution of the reaction partner immersed or covered with this. By adhesive forces in this case, the carrier is covered by a layer of the reactant.

As an immunological reaction partner, which is applied to the carrier antigens or antibodies, e.g. nucleotides, proteins, Glycoproteins, lipoproteins, glycolipids. These can be tissue or blood different animal species, bacteria, fungi or viruses. Antibodies can be formed by administering such substances to animals or occur spontaneously in body fluids of animals. You react specifically with the substances listed above, for example with substances such as DNA, actin, histone, Immunoglobulins.

These first immunological reactants are to be determined Suitable of substances that are usually found in body fluids in the event of illness occurrence. The second reaction partners are essentially components of the blood plasma or other body fluids or microorganisms occurring in them, from this resulting antigens or their metabolic products.

It is within the meaning of the invention that a third immunological reaction partner which is used against the second reactant to be analyzed is directed and is provided with a marking agent. As a third reactant come preferably directed against the immunologically reactive material to be determined specific antibodies in question. These are with regard to those listed as examples The following antibodies or anti-serum preparations are substances to be determined: Native Antibodies, e.g. preparations against human immunoglobulins or against others Serum proteins, antibodies against microorganisms, antigen or metabolic products of microorganisms, furthermore cleavage products of antibodies, which with the to analyzing partners enter into a connection, such as F (ab) 1 or F (ab) 2 cleavage products of immunoglobulin molecules.

Such antibodies are produced by vertebrates, preferably for this purpose commonly used experimental animals such as rabbits, goats, mutton, beef or Horse, by immunization with the relevant antigen and obtaining the blood serum won.

As already mentioned at the beginning, the last one introduced into the reaction third reaction partner m4t a Flarkierungsmittel for its detection and / or provided for its determination. For radioactive labeling, for example the procedures mentioned in the following work in question: W.M. Hunter, British Medical Bulletin, Volume 30, Pages 18-23 (1974) "Pre2aration and assessment of radioactive tracers ". For enzyme labeling, for example, the following work methods mentioned are used: G.B.Wisdom, Clin. Chemistry, Volume 22, No. 8, page 243 (1976) "Enzyme Immunoassay".

Methods are used for marking with fluorescent substances described by W. Page Faulk, H. Hijmans, Progr. Allergy, Vol. 16, pages 9-39 (1972) and by G. Wick, S. Baudner, F. Herzog, Immunfluoreszenz, Medizinische Yerlagsgesellschaft, Marburg / Lahn, pages 44-46, (1976). Of the numerous for the fluoroimmunoassays known fluorescent substances are preferably used for the purposes of the invention Pluorescein isothiocyanate (FITC) and tet ramethyl rhodamin- i sothiocyana t (TRITC>.

In addition to the methods described, the special subject of present invention an apparatus for carrying out the method. She is marked by a water-insoluble carrier which is indifferent to the reaction a flat surface which is coated with an immunological reaction partner is.

Suitable carriers are described above; homo- and copolymers of methyl methacrylate are preferred. These homo- and copolymers, which are particularly suitable for fluorescence immuno-analyzes due to their low intrinsic fluorescence, have the following structural components: These elements, alone or together with the structural elements of one or more monomers suitable for copolymerization with methyl methacrylate, such as acrylonitrile, styrene, vinyl chloride, vinylidene chloride, vinyl ethers, acrylates or methacrylates and acrylic acid or methacrylic acid, can make up the carrier material.

The supports are made by known polymerization methods, e.g. radical or ionic polymerization prepared and in a known manner, e.g. processed by extrusion or injection molding.

Taking into account the method according to the invention for analytical Determination of immunological reaction partners, it is appropriate to the claimed Device and the other reagents, in particular that for detection and determination of the second reactant necessary reagent provided with the marking agent (third Reakeqor partner), to be summarized in a test system. Such a thing Test system, which consists of one or more of the claimed devices and in a common outer packaging the reagent provided with the marking agent Contains, as well as its use, is to be counted as part of the subject matter of the invention.

The invention is explained in more detail by the following examples: example 1 1. Activation of a film made of poly (methyl methacrylate) A 50 µ thick film (80 x 250 mm> from polymethyl methacrylate (pMZ1A) is 15 minutes at 90 ° C with hydrazine hydrate Treated in excess, rinsed in methanol with 28 acetic acid and then with Water washed.

The slide is in a solution of 1,000 ml of ice water and 500 ml 1 molar hydrochloric acid is added to 120 ml 1 molar sodium nitrite solution. After 15 Minutes, the film is washed neutral with ice water and lyophilized.

Covalent bond between film and immunological reaction partner Human IgG is attached to the activated polymethyl methacrylate fleece by immersion into a 1% solution of the protein in phosphate-buffered saline pH 8.3 (PBS 8,) for 24 hours at 40C. Then the film is removed from the solution taken out, washed three times with the buffered saline solution. After drying At room temperature, the film is used for the immunological determination of an anti-IG Antibody available.

Determination of the anti-Igd The film is in several dilutions of goat anti-human IgG starting from a 1% strength Solution, held.

Excess anti-human IgG is removed by washing three times with PBS 7.0 removed. The film is then treated with a solution for 2 hours at 200C (0.34% w / v of protein) a rabbit anti-goat serum which conjugated with fluorescein isothiocyanate (FITC). To During this incubation period, the foils are repeatedly treated with PBS or with distilled Rinsed with water. The evaluation is carried out quantitatively by determining the fluorescence strength with an appropriate microscope (AXIOFAT from ZEISS).

The results are given in Table 1 below: Ta b e 1 1 e 1: m = mean value s = standard deviation compared to a standard uranylactete A-H IgG IgG m s m s 1: 1 163.5 10.7 140.9 7.3 1: 10 159.3 10.8 121.0 5.4 1: 100 143.7 5.8 108.0 4.7 1: 1000 123.9 7.8 101.4 2.9 1: 10,000 108.6 3.2 103.0 2.9 It could thus be shown that antibodies against human immunoglobulin G im Goat gamma globulin up to a sensitivity of 10 - 100 nanog / ml (dilution 1: 10,000) could be detected.

Example 2 Adhesive winding between carrier and immunological reaction partner Glass slides degreased in acetone are treated at 200C with a 1% gene which contains 2t bovine serum albumin, overlaid. The proteins were buffered in phosphate Saline solution of pH 8.0 dissolved.

After 24 hours, the coated slides are buffered with phosphate Saline solution of pH 7.0 washed three times and then freeze-dried. After Drying stands the slide for the immunological determination of one against human gamma globulin directed antibody available.

Determination of the anti-human gamma globulin at previously marked locations of the slide coated with the human gamma globulin become different Dilutions of rabbit anti-human immunoglobulin and normal as a control Rabbit immunoglobulin, both: conjugated with flucrescent isothiocyanate (FITC), applied and incubated for 30 minutes at 200C. After washing the incubated three times Slide with phosphate-buffered saline at pH 7.0 becomes the slide also conjugated with FITC with goat anti-rabbit gamma globulin, in a dilution of 1:40 and incubated again for 30 minutes at 200C.

After washing again with phosphate-buffered saline from pH 7.0 the slides are air dried in a microscopic device for quantitative immunofluorescence (AXIOSET from ZEISS) the fluorescence strength determined in a specified section. The result is in the table below recorded: Table 2 There are mean values (m) and standard deviations (s) of the Fluorescence shown in comparison to a standard uranyl acetate.

Anti-H IgG Kan. IgG a m + s 1: 1 16.3 2.7 19.4 1.6 1: 10 28.9 1.8 14.3 1.2 1: -100 21.3 1.9 13.0 0.8 1: 1000 14.7 0.5 13.8 0.8 1: 10,000 13.4 0.5 12.6 0.5 blank value 10.5 + 0.5 It was thus possible to detect specific Antibodies to human immunoglobulin are shown in rabbit garmaglobulin.

Example 3 Adhesive bonding of an immunological reaction partner and a film made of polymethyl methacrylate ~~~~~ A polymethyl methacrylate film, as used in example 1, is for 24 hours at 4 0C in a 1% solution of deoxyribonucleic acid in trishydroxymethylamino: methane-buffered saline The film is then washed three times with the buffer mentioned and at Room temperature dried.

Determination of deoxyribonucleic acid antibodies The DNA-coated Slide is dated for three hours at 200C with an anti-DNA serum. Washing in contact brought. Rabbit normal serum is used as a control. After incubation is done three times washed with phosphate buffered saline, pH 7.0. Then takes place an incubation with a fluorescence isothiocyanate-labeled anti-rabbit gamma globulin from the goat at a dilution of 1:20 for 30 minutes at 200C. In connection the film is then treated three times with phosphate-buffered saline solution of pH 7.0 and washed once with distilled water and then dried. In a microscopic The device for quantitative immunofluorescence (AXIOMAT from ZEISS) measures the fluorescence strength determined in a section of the film.

The result is shown in Table 3. It shows the fluorescence value (m) with the associated standard deviation (s) compared to a standard uranyl acetate: T a b e 1 1 e 3: m s Anti-DNA 58.4 3.6 Can.-NS 37.6 1.4 Blank 30.4 1.6

Claims (10)

n Immunological analysis method PATENT ANSPJ <ÜCHE 0 Immunolocjisches Analytical method with a carrier-bound immunological reaction partner, thereby characterized in that a water-insoluble one which is indifferent to the reaction Tzäqex, who coated one with a first immunological reaction partner has a flat surface, with one containing a second reactant Solution is brought into contact, after a sufficient reaction time the contact canceled and the reactants trager.-de carrier with a third one Marking agent tray end reaction partner is brought into contact, its immunological Specificity is directed against the second reactant, after which the amount of bound marking agent is detected or determined. 2. The method according to claim 1, characterized in that the carrier is a homo- or copolymer of methyl methacrylate. 3. The method according to any one of claims 1 and 2, characterized in that that the carrier is a film of 10-100 thickness. 4. The method according to any one of claims 1 to 3, characterized in that that the carrier with the first immunological reaction partner through a covalent Bond is linked. 5. The method according to any one of claims 2 to 4, characterized in that that the carrier is treated with hydrazine hydrate before coating and that at the same time The hydrazide formed on its surface is converted into the acid azide. 6. The method according to any one of claims 1 to 5, characterized in that the labeling agent is a fluorescent substance. 7. Application of a method according to any one of claims 1 to 6 for the quantitative determination of immunological reaction partners in a body fluid. 8. Device for performing a method according to one of the Claims 1 to 6, characterized by an indifferent water-insoluble carrier with a flat surface which is coated with an immunological reaction partner is. 9. Apparatus according to claim 8, characterized in that the carrier a film of a homo- or copolymer of methyl methacrylate of 10-100 LL thickness is. 10. Device according to one of claims 8 and 9, characterized in that gelsennzeichr.et, that the ixmunologsscile reactant with the carrier through a covalent bond is linked.