DD158676A5 - PROCESS FOR THE IMMUNOLOGICAL DETERMINATION OF ENZYMES, MEANS FOR IMPLEMENTING THE METHOD AND ITS USE - Google Patents

Abstract

The invention relates to methods and means for the immunological determination of enzymes which form an enzyme-inhibitor complex with an inhibitor, in particular for the determination of proteases. The method for the determination of enzymes by means of antibodies bound to a water non-soluble carrier against the enzyme to be determined is characterized in that enzymes which form an enzyme-inhibitor complex with an inhibitor are incubated with the bound antibodies, the sample solution is removed, the washed-out, loaded with the enzyme-inhibitor complex antibody carrier with a coupling product formed from a specifically reacting with the inhibitor antibody and a marker substance incubated, the unbound coupling product removed and the concentration of the marker substance on the solid phase as a measure of the determined enzyme concentration determined.

Description

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Merck Patent Limited Liability Company

Darmstadt

"Method for the immunological determination of enzymes, means for carrying out the method and its use

Field of application of the invention

5- The invention "relates to methods and means for the immunological determination of enzymes which form an enzyme-inhibitor complex with an inhibitor, in particular for the determination of proteases.

Characteristics of the "known technical solutions" In inflammatory processes, proteases from poly-morphaminous leucocytes and macrophages play a causal role in the damage of the affected tissues.The leukocytic proteases such as elastase or collagenase are localized in the lysosomes of the white blood cells and become pathological Stimuli are released into the extracellular milieu, where they cause a breakdown of connective tissue substances and, after entry into the bloodstream, a disturbance of the homeostasis eg by degradation of coagulation factors and fibrinogen.The quantitative detection of leukocytic proteases in the blood is therefore very valuable for the assessment of the acuteness of Inflammation, for the follow-up of chronic inflammation, for the control of

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Therapy and prognosis. The determination of these factors appears to be necessary whenever leukocytosis occurs in connection with diseases, such as "certain infectious diseases, intoxications, tissue necrosis, endocrinological diseases, malignant tumors

The exact quantitative detection of leukocyte proteases in the blood has hitherto hindered by the fact that these proteins on entering the circulation rapidly complexes with the protease inhibitors which are abundant in the blood, for example with .alpha.] -Anti trypsin, .alpha.-p-macrognum globulin and others, form. The complexes with α, --antitrypsin are stoichiometrically constructed compounds of one molecule of protease and one molecule of inhibitor, forming a covalent bond. In the complex, the protease is enzymatically inactive. Therefore, the provision of. "enzymatic activity completely unsuitable for the quantitative detection of the protease concentrations in plasma, serum or in other body fluids _w ie synovial fluid, exudate, tissue extract, etc.

Elastase (EC 3.4.21.11) is of particular importance among the leucocyte proteases: it is abundant in the leucocytes and, together with collagenase, accounts for 5 % of the dry weight of granulocytes.

The enzyme has a low substrate specificity and can proteolytically degrade many different biologically important substances, such as elastin, proteoglycans, collagen, IgG, complement factors, fibrinogen and coagulation factors. Elastase in plasma is 92% bound to o-antitrypsin and too 8 % bound to other inhibitor molecules Complexes of elastase and α, - anti trypsin were detected in inflammatory processes eg in plasma, serum, peritoneal fluid and pus.

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Since the proteases are always bound to inhibitors in body fluids, their detection is practically only possible with the aid of immunological methods. Well-known methods are the electroimmune diffusion, the radioimmunoassay and the agglutination tests or agglutination inhibition tests. However, these methods have considerable disadvantages. Electro-immunodiffusion is tedious and inappropriate for routine use; the evaluation is only qualitative and poorly reproducible. Radiol immunoassay is also time consuming and disadvantageous because of the use of radioactive substances. Particular disadvantages are: necessity of special laboratory equipment, special safety measures due to the radiation hazard, low stability of the radiolabeled reagents, disposal, etc.

From DOS 26 41 840 an agglutination test is known in which the plasma to be examined with a suspension of particles of synthetic resin or blood cells carrying antibodies against an enzyme-inhibitor complex on its surface, contacted and then observed directly whether Agglutination occurs or not. This method has the disadvantage that it lends only semiquantitative results and is not routable. The antibodies used must be made specific by elaborate methods of preparation for antigenic determinants that are present only in the enzyme-inhibitor complexes.

DAS 22 06 103 describes a method in which a component to be determined reacts with an insolubilized binding partner and one of these two partners is detected via an enzyme-labeled indicator molecule. The specific quantitative determination of an enzyme-inhibitor complex is not possible by this method. OBJECT OF THE INVENTION The present invention is based on the object, methods and means for the quantitative determination of

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Enzyme, which are in the form of enzyme-inhibitor complexes, to provide, with which the described disadvantages of the known methods can be avoided.

Explanation of the essence of the invention;

According to the invention the Aul'gäbe was solved by> that one binds an enzyme-inhibitor complex using a solid-phase antibody against the enzyme moiety "and the determination of a labeled antibody against the inhibitor portion of the Molekularverbandes.unterfolge.

The invention relates to a method for the determination of enzymes by means of antibodies bound to a water-insoluble carrier. Against the enzyme to be determined, which is characterized in that enzymes which form an enzyme-inhibitor complex with an inhibitor with the incubated antibodies, the sample solution removed, the washed-out, loaded with the enzyme-inhibitor complex antibody carrier with a coupling product formed from a specifically reacting with the inhibitor antibody and a marker substance incubated, the unbound coupling product removed and the concentration of the marker substance determined on the solid phase as a measure of the enzyme concentration to be determined.

Furthermore, the invention relates to means for carrying out this method, containing antibodies coated against the enzyme to be determined coated reaction vessels and a coupling product formed from a specific reacting with the inhibitor antibody and a Markersübstanz. Another object of the invention is the use of these agents for the determination of enzymes which form an enzyme-inhibitor complex with inhibitors.

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With the method and means according to the invention, in principle all enzymes can be determined which are able to form an enzyme-inhibitor complex with an inhibitor and are therefore inaccessible to direct enzymatic activity determination. The method is particularly suitable for the determination of complexes of proteases with the respectively relevant protease inhibitors for them, such as elastase / cCj-antitrypsin, thrombin / anti-thrombin III, plasmin / antiplasmin, trypsin / - ^ antitrypsin etc. The method is specific, simple, fast and sensitive. Only three reagents specially prepared for this purpose are required (antibody-coated reaction vessels, enzyme-labeled antibodies, solution with a defined concentration of the enzyme-inhibitor complex), and this is possible with the equipment of a normal clinical chemistry laboratory. The method is based on the specific binding of the enzyme-inhibitor complex to immobilized antibodies to the enzyme and a subsequent quantitative determination of the bound complex with labeled antibodies to the inhibitor. For detection, therefore, two antisera with absolutely different binding specificity, specifically for one of the two partners of the molecular association, are used.

One for the e.g. proteolytic enzyme-specific reactant present in the protease-protease inhibitor complex is bound to a solid support. Reactants specific for the protease are antibodies or their antigen-binding fragments. Specific antibody preparations are isolated from the sera of specifically immunized animals. As immunogen, the protease is in

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inhibited state after reaction with low molecular weight inhibitors or used in untreated state. Suitable experimental animals for the production of the antisera are, for example, sheep or rabbits, but other experimental animals are also suitable.

The solid phase antibody to the protease may be free of carrier, e.g. be made insoluble by crosslinking with polyfunctional reagents such as dialdehydes, dioxides, etc., but preferably the antibody is bound to a solid support material. The solid support may consist of any material inert to the substances used in the assay to which antibodies to proteases can be bound. Suitable carriers are e.g.

Shaped bodies such as amorphous particles, spheres, platelets, foils or reagent vessels made of inorganic material (eg glass) or organic material (eg homopolymers or copolymers of vinyl compounds such as olefins, vinyl acetate, vinyl chloride, vinylidene chloride, tetrafluoroethylene, styrene, acrylic acid or methacrylic acid, furthermore polymers of Formaldehyde and cyclic acetals as well as polycondensates such as polyesters, polycarbonates or polyamides or crosslinked carbohydrates and crosslinked polypeptides), furthermore biological particles such as cells (eg erythrocytes).

The binding of the antibodies to the solid support may optionally be adsorptive, adhesive or covalent. It is possible to fix the antibodies to solid supports by conventional methods of protein chemistry for covalent attachment of biologically active polypeptides. Typical examples of this are the activation

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support surfaces by the cyanogen bromide method or surface crosslinking of the antibodies on the support by polyfunctional crosslinking agents such as glutaric dialdehyde and the like.

It has been found that with numerous carrier materials, the antibodies to the protease can be sufficiently firmly bound to the surface of the carrier by adsorption or adhesion. For the coating, the carrier is contacted with the antibodies or their antigen-binding fragments. For example, the protein is dissolved in an aqueous solution, preferably a 0.15M saline or phosphate buffered isotonic saline (= PBS) pH 7.2, in a protein concentration of 1μg to 1g / L, preferably

1-5, 10 mg / 1, dissolved. The protein solution is prepared by diluting the blood plasma or eluate of specifically immunized animals, but preferably by dissolving purified immunoglobulin from the antiserum. The solution is allowed to stand for 1 minute to 5 days, preferably 2 hours, with the carrier. Thereafter, the solution is separated from the carrier, for example by pouring from the container; the carrier is washed with buffer, conveniently containing a detergent (e.g., PBS pH 7.2 with 0.05% polyoxyethylene sorbitan monolaurate).

The determination of the protease-protease inhibitor complexes is carried out in such a way that the carrier which has been coated with specific antibodies against the protease to be determined or with the antigen-binding fragments from the antibodies, 1 min to 48 hours, preferably

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2.hours, with the liquid containing the protease-protease inhibitor complexes, ζ. Β '. Serum sample, is incubated. In parallel runs, samples of known concentration of the protease-protease inhibitor complexes are used. These samples are prepared by diluting a reaction mixture of highly purified protease and highly purified protease inhibitor in buffer (eg PBS pH 7.2) or in human plasma previously inactivated at 56 C for 30 min.

After completion of the incubation, the solution is separated from the carrier, the carrier is conveniently washed with buffer (e.g., PBS pH 7.2) to remove excess sample components. Subsequently, the thus treated support is incubated with a solution of a labeled second reactant specific for the complexed protease inhibitor. The duration depends to some extent on the conditions in terms of pH, temperature and concentrations. In general, an exposure time of 2 to 24 hours. Reactants for this experimental step are antibodies or antibody fragments directed against antigenic determinants of the complexed protease inhibitor molecule. Specific antibody preparations are isolated from the sera of specifically immunized animals. It has proven to be expedient to enrich the antibody fraction from these antisera by methods of immunoadsorption on immobilized antigen, antigen-specific and to separate from antibodies of unknown specificity.

As a marker for the second reactant, e.g. Enzymes, luminescence or electron density markers,

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used fluorescent or radioactive substances. Within the scope of the invention, preference is given to using those marker enzymes whose activity can easily be determined by an optical test, in particular by a color reaction in visible light or by a change in the concentration of NADH or NADPH. Typical examples of suitable labeling enzymes are alkaline phosphatase, peroxidase, glucose. oxidase, £ *, and β-galactosidase, glucoamylase, invertase, malate dehydrogenase or glucose-6-phosphate dehydrogenase. Covalent bonds of the respective marker enzymes with the specific antibodies against protease inhibitors or antigen-binding antibody fragments are carried out according to methods known from the literature / BZ Histochem Cytochem., 22, 1084 (1974); Bull. Soc. Chim. Biol. 50, 1169 (FIG. 19682 / ·

The optimal dilutions of the labeled antibodies against the complex-bound protease inhibitor to be used in the test are determined by preliminary experiments. They must not be nonspecific, i. to form detectable compound with the carrier without the presence of carrier-bound protease-protease inhibitor complex. For this purpose, the solution of the labeled antibody is a detergent, e.g. 1 mM sodium dodecyl sulfate added.

After completion of the incubation, the solution is separated from the carrier, for example by pouring from the container; the carrier is washed several times with buffer, which suitably contains a detergent, and then the amount of label-bound carrier is determined by conventional methods. To control for unspecific binding of labeling agent on the carrier are experiments that, as described above, but with

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Samples without protease protease inhibitor complex of the composition to be determined are performed.

The content of protease-protease inhibitor complex with the protease to be determined in a biological sample is quantified by comparison with the standard samples described above.

The compositions of the invention may additionally contain conventional stabilizing agents such as protein additives (bovine serum albumin or ovalbumin), carbohydrates, glycerol, bacteriostats, fungicides, etc.

The invention is further illustrated by the following examples.

Examples 1example 1

Quantitative immunological detection of complexes of leukocyte elastase and α-antitrypsin

a) Preparation of antiserum against leukocyte elastase

Elastase was recovered from human polymorphonuclear leukocytes and purified by affinity chromatography and ion exchange chromatography to such an extent that no contaminating other protein was detectable in polyacrylamide gel electrophoresis at pH 4.3 and in immunoelectrophoresis with human serum protein antiserum. The freeze-dried, salt-free protein was dissolved in 0.15 M saline at a concentration of 2 mg / ml. 1 ml of the solution was mixed with 1 ml

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complete friend ¹ see adjuvant emulsified. This emulsion was injected to sheep or goats at 3 to 4 places in the hind legs. Same injections were made twice at intervals of three weeks each. Blood was collected 21 days after the last injection. Further injections were made 12 weeks after the last blood sample. Blood was taken ¹ 14 days after the booster injection. The serum was obtained by conventional methods from the blood, stabilized by the addition of 0.05% sodium azide and 0.05% Natriumäthylquecksilberthiosalicylat and stored at -20 C.

b) Preparation of antiserum to cCj-antitrypsin

The antigen used is highly purified OL ^ -antitrypsin from human plasma. Antiserum to OC - antitrypsin was prepared in sheep or goats following the same schedule as described in a). For the immunization of rabbits, 1 mg of freeze-dried, salt-free α-antitrypsin was dissolved in 0.5 ml of 0.15 M saline. The solution was seen with 0.5 ml complete Freund's adjuvant ¹ emulsified and the emulsion was injected into the animal at 2 to 3 places in the hind legs. A similar emulsion was made 4 weeks later sc in the neck. 7 days later, about 40 ml of blood was withdrawn from the marginal ear vein. 4 weeks later there was another booster injection sc and a blood sample taken 7 days apart. The serum was treated as described in a).

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c) isolation of immunoglobulin from the antisera

The IgG fraction from the antisera against elastase or against OC ₁ -antitrypsin was by conventional methods / Scand. J. Immunol. 2_, Suppl. 1, 161 (1973) or Arch. Biochim. Biophys. 134 , 279 (1969) 7 isolated ^from the sera and stored in salt-free, freeze-dried form.

d) Purification of Antibodies to OCj-Antitrypsin by Immunoadsorption

Antibodies to the protease inhibitor were immunospecifically isolated by chromatography of purified immunoglobulins (according to c)) from antisera to OC ₁ -antitrypsin (to b)) via Sepharose 4B or via substituted agarose containing covalently bound CL 4 -antitrypsin. Antibodies to the protease inhibitor remained attached to the immobilized antigen and were eluted with 0.1 M glycine / HCl buffer pH 2.8 or 3 M potassium thiocyanate solution. Approximately 5 to 20% of the given immunoglobulin was immunized in this way.

20 · specifically isolated; Immunoglobulins with a different specificity were thus removed. The eluate with the specific antibodies was dialyzed against phosphate-buffered saline and then against 0.01 M ammonium bicarbonate solution. Subsequently, the protein was freeze-dried.

e) Preparation of Enzyme-Antibody Conjugates

Immunospecifically purified antibodies (after d)) were cross-linked with calf-intestinal alkaline phosphatase using 0.2% glutaric dialdehyde / Biochim. Biophys. Acta 251 , 427 (1971)

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was chromatographed on a column of Bio-Gel A 1.5 m (26 mm × 900 mm). The enzyme-antibody complexes were included in the first peak of the eluate's AP activity. The solution was stabilized by the addition of ovalbumin (ad 0.1%) and sodium azide (ad 0.05%) and stored at 4 ° C.

f) Coating of reaction vessels with antibodies against elastase

Antibody to elastase prepared according to a) and purified according to c) was immobilized by adsorption on plastic reaction vessels. Reaction tubes made of polystyrene 12 χ 55 mm, round cuvettes or plates for microtitration were used as reaction vessels. In the typical approach, the tubes or cuvettes were filled with 1 ml of an IgG solution containing 10 μg IgG / ml (in 0.15M saline containing 0.02% sodium azide). For the plates, 0.2 ml of a 50 μg / ml IgG solution was used per depot. The vessels were sealed and allowed to stand overnight at 4 ° C. Before use in the test, the liquid was decanted or aspirated. The tubes were then washed several times with rinse solution (0.15 M saline with 0.05% detergent and 0.02% sodium azide) and briefly air dried.

g) Preparation of complexes of leukocyte elastase and OC ₁ -antitrypsin

As an analytical sample for quantitative detection, the complex of protease and protease inhibitor was generated in vitro by incubation of the two isolated highly purified components. To this was added 0.2 ml

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of a solution of 0.5 mg of pure leukocyte elastase (human) in 1 ml (dissolved in 10 mM Tris / HCl buffer pH 7.4 with 0.12 M sodium chloride) and 0.2 ml of a solution of 2.8 mg pure Ct - antitrypsin (from human plasma) in 1 ml (dissolved in 50 mM Tris / HCl buffer pH 8.8 with 0.05 M sodium chloride) and then incubated for 2 hours at 37 ° C. The mixture was then stored at -20 ° C.

Analysis by immunoelectrophoresis showed that all the elastase in the incubation mixture was converted to the elastase / oCj-antitrypsin complex.

h) Recovery of complexes of leukocyte elastase and cc. antitrypsin

In microtiter plates coated with antibodies against elastase according to f) and prepared for use, 0.2 ml of sample containing 0.1-10 ng elastase in the inhibitor complex per depot were filled. The samples with the defined elastase content were obtained by diluting the enzyme-inhibitor mixture according to g); The diluent was phosphate-buffered saline. To determine blank values, several depots were filled in parallel with 0.2 ml of diluent without enzyme-inhibitor complex. After incubation for 2 hours at room temperature, the contents of the depots were aspirated. After washing three times with phosphate-buffered saline, solution with enzyme-antibody conjugate was added to e) (0.2 ml per depot). The solution was previously adjusted to about 800 U / l AP activity. Diluent therefor was 10 mM Tris / HCl buffer pH 7.5 with 2 mM magnesium chloride, 0.025 mM zinc chloride, 1% ovalbumin, 1 mM sodium dodecyl sulfate and 0.02% sodium azide. The plates were air-

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sealed and incubated overnight at room temperature. Afterwards the liquid was sucked off. The plates were rinsed three times and briefly air dried. Thereafter, buffer substrate solution for the determination of alkaline phosphatase activity was filled in a staggered sequence at 0.2 ml per depot. After 10 min. Incubation at room temperature, the reaction mixture was transferred from the depots in a microcuvette and measured the extinction. The zero value was buffer substrate solution. Depots without added protein gave an absorbance, which is a measure of the non-specific adsorption of enzyme-antibody conjugates to the depots. In depots containing protease-protease inhibitor complexes, increased absorbance values were found. For evaluation, the extinction found (minus the extinction for nonspecific adsorption) was compared with the amount of elastase (ng) used in the inhibitor complex. The table below gives the final absorbance values at 405 nm after a reaction time of 10 minutes, depending on the amount of elastase.

Elastase / _nc [/ OD _405/10 min 12.5 1, 000 6.25 1, 030 3.125 0,830 1, 563 0,540 0.781 0.315 0.390 0.115 0.195 0,030 0 = blank value 0

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The data show that the concentration of elastase OL-antitrypsin complexes can be quantified in this way. There is a strongly correlated relationship between the amount of about 0.4 ng to about 6 ng of complexed elastase in the sample and the extinction measured. The buffer-substrate solution used was a solution which had the following concentrations tested: 10 mM p-nitrophenylphosphate, 0.5 mM magnesium chloride, 1.0 mM diethanolamine rICl buffer pH 9.8.

Example 2

Quantitative immunological detection of complexes of thrombin and antithrombin III

a) Isolation of immunoglobulin from the antisera

As a source of the specific antibodies against protease and protease inhibitor, commercially available rabbit antisera against human prothrombin or against antithrombin III from human plasma were used. The IgG fractions from these antisera were isolated by known methods and stored in salt-free freeze-dried form.

b) Preparation of Enzyme-Labeled Antibodies to Human Antithrombin III

The immunoglobulin fraction from antiserum to human antithrombin III isolated according to a) was chemically covalently linked to the marker enzyme alkaline calvatase from calf small intestine, and the conjugates of IgG and enzyme prepared in this way were further processed analogously to Example 1e).

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σ) coating of reaction vessels with antibodies against prothrombin

The purified according to a) immunoglobulin fraction from the antiserum to human prothrombin was immobilized by adsorption on plastic vessels. For this purpose, polystyrene microtiter plates were used, which were filled with 0.2 ml of a solution of 50 yiq immunoglobulin per ml (in 0.15 M saline solution containing 0.02% sodium azide) per depot. The plates were covered airtight and allowed to stand overnight at 4 ° C. Before use in the test, the IgG-containing fluid was removed. The vials were washed several times with 0.15 M saline containing an additional 0.05% detergent and 0.02% sodium azide and briefly air-dried.

d) Preparation of molecular complexes of human thrombin and human antithrombin III

The complexes of human thrombin and antithrombin III for use as an analytical sample in the assay were generated by incubation of mixtures of the two components in highly purified form. For this purpose, 50 units of antithrombin III (= heparin cofactor) and 50 units of heparin in a volume of 1.26 ml in 10 mM Tris / HCl buffer pH 7.5 were incubated at 37 ° C for 30 minutes and then the mixture 50 units of thrombin (corresponding to 16.7, ug enzyme protein) was added in 50 ul _y · of the same buffer. After incubation for 2 hours at 37 ° C, the reaction solution and appropriate dilution steps were used in the test.

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e) Quantitative detection of thrombin antithrombin. III complexes

In the depots of microtiter plates coated according to c) with antibodies against prothrombin and prepared for the test, 0.2 ml of sample with known content of thrombin in complex-bound form were filled. As samples of known content, dilution steps of the incubation mixture according to d) in phosphate-buffered saline were used. To assess blank values, some depots were filled with 0.2 ml of diluent without enzyme-inhibitor complex. After 2 hours incubation at room temperature, all depots were emptied and washed three times with phosphate-buffered saline, which additionally contained 0.05% detergent. Thereafter, 0.2 ml of solution with enzyme-labeled antibodies against antithrombin III, which were prepared according to b), were introduced per depot. The solution was previously adjusted to about 300 U / l AP activity. The diluent used was 10 ml Tris / HCl buffer pH 7.5, which additionally contained 2 mM magnesium chloride, 0.025 mM zinc chloride, 1% ovalbumin, 1 mM sodium dodecyl sulfate and 0.02% sodium azide. The plates were sealed airtight at room temperature overnight. Thereafter, the liquid was sucked off. The plates were rinsed three times and briefly air dried. Then 0.2 ml buffer substrate solution was filled per depot for the AP test. After a 20 min incubation period, the reaction mixture from the depots was transferred to a microcuvette and the absorbance was measured in the photometer. The zero value was buffer substrate solution.

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Increased absorbance values were found in depots containing protease protease inhibitor complexes. For evaluation, the extinction found at 405 nm in the buffer substrate solution after 20 min incubation in the respective depots of the calculated amount of complex-bound thrombin used compared.

The table below gives the final absorbance values at 405 nm after 20 min of reaction depending on the amount of antigen.

Thrombin / ng / ^OD 4O5 ^{/ 20 min} 1270 0,400 635 0.390 318 0.321 159 0,279 79.5 0.196 39.8 0,137 19.9 0,099 10.0 0.058 5.0 0,025 0 = blank value 0

The data show that there is a strictly correlated relationship between the amount of antigen used and the photometrically measured enzymatic indicator reaction, which allows a quantitative determination of thrombin-antithrombin III complexes.

Claims (6)

229527 2 invention claim 1. A method for the determination of enzymes by means of bound to a water-insoluble carrier antibodies to the enzyme to be determined, characterized in that enzymes which form an enzyme-inhibitor complex with an inhibitor, incubated with the bound antibodies, the sample solution is removed , the washed-out, loaded with the enzyme-inhibitor complex antibody carrier with a coupling product formed from a specific reacting with the inhibitor antibody and a marker substance incubated, the unbound coupling product removed and the concentration of the marker substance on the solid phase as a measure of determines the enzyme concentration to be determined. 2. The method according to item 1, characterized in that the enzymes to be determined proteases and the inhibitors are protease inhibitors. 3. The method according to item 1 and 2, characterized in that the enzyme to be determined is elastase from leukocytes and the inhibitor ot.-antitrypsin. 4. Means for carrying out the method according to points 1 to 3, characterized in that it contains antibodies coated against the enzyme to be determined coated reaction vessels and a coupling product formed from a specifically reacting with the inhibitor antibody and a marker substance. - If- 22 952 7 2 5. Composition according to item 4, characterized in that it contains reaction tubes coated with antibodies against proteases and a coupling product formed from an antibody specifically reactive with the protease inhibitor and a marker substance. Composition according to items 4 and 5, characterized in that it contains reaction vessels coated with antibodies against elastase from leukocytes and a coupling product formed from an antibody specific for c ^ -antitrypsin and a marker enzyme.