CS219900B2 - Method of immunological determination of the enzyme and means for executing the same method - Google Patents

Abstract

1. Method for the determination of enzymes with the aid of antibodies against the enzyme to be determined, which are bonded to a water-insoluble carrier, characterised in that enzymes which, with an inhibitor, form an enzyme/inhibitor complex are incubated in form of such a complex with the bonded antibodies, the sample solution is removed, the washed antibody carrier which is charged with the enzyme/inhibitor complex is incubated with a coupling product formed from an antibody, which reacts specifically with the inhibitor, and from a marker substance, the coupling product which has not been bonded is removed and the concentration of the marker substance on the solid phase is determined, as a measure of the enzyme concentration which is to be determined.

Description

The invention relates to a method and composition for the immunological determination enz ^y MU, which forms a complex with an inhibitor of the enzyme-inhibitor, especially ^p ro ^y tických stanovern proteolytic enzymes.

In inflammatory processes, proteases from polymorphic leukocytes and macrophages play an essential role in damaging the tissue present. Leukocyte proteases such as elastase or- collagenase, .jsou localized in lysosomes and white blood cells after pathological irritation are secreted into extracelulárrnho free of ^'r of ^copper. Supplied from ^d eo ^d bourávání tissue binding substances, and after entry of blood DO- ^run at způsobuj homeostasis disorders e.g. degradation of fibrinogen and clotting factors. Quantitative evidence of leukocyte proteases in the blood is very valuable for assessing akutity Z ^and n tlivého ^{of Pro-p} Observe a disease ^and the course of chronic inflammations, for therapy control and for forecasting. · The determination of these factors, ^as a phenomenon particularly necessary when the disease occurs .souvislosti with leukocytosis, as-for example, when certain -infekčních diseases, intoxication, tissue necrosis, endocrinological -onemocněních, malignant tumors and the like.

The exact quantitative evidence of leukocyte proteases in the blood was still hampered by the fact that these proteolytic enzymes in the blood entering · - circulation rapidly forms complexes with inhibitors · ^p where ^E roteolytických enzymes are well represented in the blood, such as alpha-antitrypsin, -alfa2- macroglobulin and the like. ^The OM ^{pl y} ex alpha-antl'tr hoot ^y are stoichiometric compounds built from one molecule ^{yp y} roteol ^Star tick it enz ^y him and one ^e inhibitor molecule to form a covalent bond. In said complex, the protease is enzymatically inactivated. For tohoto- reason tent ^as enzymatic ^and oven. ACTIVITIES NOT SUITABLE FOR THE QUANTITATIVE DETERMINATION OF PROTEOLYTIC ENZYMES CONCENTRATION IN Plasma, serum or else in other body fluids such as synovial fluid exudate, tissue extract and the like.

For proteolytic enzymes has Leukocyte elastase (EC 3.4.21.11) of particular importance: In a significant number -se present in leukocytes and constitutes together with Koll enázou ^g of 5% su'šin ranulocytů ^YG. This enzyme has a low substrate specificity and may roteotytick ^{p y} -odbourávat many different, ^and b olo ^g ^ icky important substances such as elastics napříWad ^p rote ^g l ^y kan ^y, collagen, immunoglobulin, complement factors, and fibrlnoge -srážecí factors. Elastase is Azm ^{pl of} 92% bound to alrápantitry sin ^ 8% binding ^and molecules into yin ^{é y} inhibitors. ^The omplex ^y from ^y and elastases alíapantitrypsinu were shown -in inflammatory processes e.g., plasma, serum, peritoneal fluid, pus and the like.

Since the proteolytic enzyme ^{characterized} in tětotoh tekutinách.stéle bound inhibitors, their evidence slingshot cally possible only with the aid of immunological metho ^d. Known such ^- meto ^dy electroimmunodiffusion e.g., radioimmunoassay and agglutination tesy of ^{P or} A ^de agglutination inhibition test. Uve.dené meto ^dy maj however ^{still have} considerable disadvantages. Electroimmunodiffusion is lengthy and unsuitable for routine determinations; the evaluation is only qualitative and difficult to reproduce. Radioimmunoassay is a ^{time-demanding No.} Ny is disadvantageous due to use of radioactive substances. Particular disadvantages are:

The necessity of special laboratory equipment without special ^{p p} at ečnostní -o ^of learning because not ^b e ^p ez ^whose -ozářený not ATRN stabihta ^p r ^d ioaktivně labeled substances, security and so on.

^DE-OS no. ² I6 ⁴¹⁸⁴⁰ is known aglutmační • Tesy ^p s where ^p is examined lazma UV ^{Adi d} by contact with the ^p enz ^s sus synthetic resin particles or- blood particles on whose surface is coated antibody complex enz ^y m-mhibitor ^{and p} Otom immediately observed -zda agglutination occurs or not. This method has the disadvantage that it gives only semi-quantitative results and cannot be used as a routine assay. The antibodies used need to be converted to a specific form of pro-antigenic determinants that are present only in enzyme-inhibitor complexes by expensive production methods.

In the DAS no. 22 06 103 describes a method in which reacting one determined component is anchored and a binding member těchto- -from both the connecting members are ^{co p} statutes dip mdikátoru enzyme labeled molecules. ^Sp tecfické quantitative determination com ^p lex enzyme-inhibitor Get ^y using this method is not possible.

The present invention thus solves the task of developing a method and means for determining the quantitative ^and -enz ^y him are exercised in ^{the y} skytují as enzyme-inhibitor complex, using kterého- -possible would eliminate the drawbacks of known-method.

P-Odle ^BC ^ ed ^é it marries ^- in finding this ^{y - l} UKo vytošen and ^from whom e -with ^p tex enzyme-inhibitor binds with the grounded phase antibody enzymového- share, followed by determining the dip ^p labeled ^{d s p} rotilátk ^? interceptor ^-p moiety of the molecular bond.

The object ^instance e ^d LO ^from Ene ^H s ^y nátezu Get ^y from ^para uso ^{b y} determining en'z Mu - ^{p p} dip rotilátek stanovovaného · enzyme linked Nave ^ga insoluble in ^p oral ntfsfÍý which is characterized in that the enzymes which form inhibitor enzyme-inhibitor complex, is incubated with the bound antibodies, the reaction solution was removed, washing with an antibody, sat ^y prices ^for om ^p Lex enzyme-inhibitor ^{of T} or ^is incubated with a coupling product formed from an antibody specifically reactive with inhibitor and the labeled substance -nenavázaný deprotecting the coupling product and the degree of concentration určovanéiho enz ^y it is determined at

219 90 based on the concentration of the labeled substance on the solid phase.

^Further, in ředložený in ^y n and ^l ez th ^{ka P} ro-center ^to U ^kp ROV ^and happenings of from ^para uso ^b u, which consists of a reaction vessel coated with an antibody stanovovaného enzyme and the coupling product formed from an antibody specifically reagujím inhibitor and represent ^: metal ^é substance.

Another ^P following shall be subject ^{Pla dl} marries vynMezu ^h o is the use of this composition for the determination enzypiů which constitute inhibitors of the enzyme-inhibitor complexes.

Use of ^P and Úsobí prostroďku Come to the invention can principally determine all enzymes which can form a complex with an inhibitor of the enzyme-inhibitor, and which therefore can not be · determined by direct measurement of enzyme activity. The present invention is particularly suitable for stanovern complex of proteolytic enz ^y Mu always relevant protease inhibitors such · is e.g. elastase and / ^{j-FAL antitrypsln,} thrombin / antithrombin ΙΙξ p ^l asmin ^{/ antiplasmin,} trypsmMlfai-antňrypsin like.

This way is specific, simple, quick and swift. ^P ro perform its required -only three specially prepared reagents (reaction vessel coated with an antibody, the enzyme-labeled antibody solution with defined concentration of the enzyme-inhibitor], wherein execution is possible with conventional equipment of apparatus wedge-CKO-chemical laboratories.

^B is caused based on the formation speciímké binding enzyme-inhibitor complex to the immobilized antibody and the enzyme, the following quantitative determination of bound labeled antibody complexed with the inhibitor. · For proof therefore use two different antisera with the absolute binding speccfitou tot ^Iz · spemfická for the en ^d of Olbo member ^of a molecular binding.

The specific proteolytic enzyme reactant present, for example, in the protein-enzyme protease inhibitor complex, is anchored to a solid support. Reaatanty speccfickými for proteolytic enzymes, antibodies .or · fragments thereof binding anti ^g ene. Pacific preparations of rats are circulated from siccifically immunized animals. The immunogens ^U sing ^{j í} protedytické enzymes ineffective state after reaction with low molecular weight inhibitors, or else in an unprocessed state. For example, sheep or rabbits are suitable test animals for the preparation of antisera, but other test animals are also suitable.

The solid phase antibodies of the proteolytic enzymes used can also be used without a carrier, for example, after cross-linking with polyfunctional agents such as dialdehydes, dioxides and the like, thereby rendering them insoluble. Preferably, however, the antibodies bind to a solid support material. A solid carrier can consist of a material which is inert ^to the reaction ^sl oz ^ka m-AnyMem Employment of the test and which can be an antibody against the enzyme-olytíckých connect.

^Vh of ^d Nym ^and a carrier ^or are for ^{Rikl d} and molded articles such as amorphous particles, beads, or Platelet-molds .reakčrn nátótay. from an inorganic material such as glass or organic material (e.g. homopolymers or copolymers of vinyl compounds such as olefins, vinyl acetate, vinyl chloride, vinilidene chloride, tetrafluoroethylene, styrene, aralkyl or methacrylic acid, polymers of formaldehyde and cyclic acetals as well as polycondensates such as such as polyester, polycarbonates or polyamides, or cross-linked carbohydrates and cross-linked polypeptides), and further biological particles such as cells (erythrocytes).

The binding of the antibody to the solid support may be adsorptive, adhesive, or optionally covalent. It is possible to fix the antibody to a solid support using conventional methods of peptide chemistry by covalently binding biologically active polypeptides. Typical methods include activation of the carrier surface by the cyanogen bromide method or surface cross-linking of the antibody to agents such as glutaraldehyde and the like.

It has been found that in various carriers, protease antibodies can be sufficiently tightly bound to the carrier surface by adsorption or adhesion. In coating, the carrier is contacted with the antibody or antigen-binding fragments thereof. For example, the protein extended ^p estuary in the aqueous Solutions of The advantage ^{of d} n ⁱⁿ 0. 15 M sodium chloride or odného in isotonic solution chlorotrimethylsilane setoého · puírovaném phosphate (e ^further reduced ^and no on ^{PBS) p} H ^7.2 at a concentration of 1 pg of protein 1 to 1 g / l, preferably ¹⁰ mg / l. The protein solution is prepared by diluting blood plasma or blood serum with cifically immunized animals, but preferably by dissolving the purified immunoglobulin from the antiserum. To flow Necmi state with noses ^P by the period of grinding the a ^{n e} mmuty and ^of the five top preferably for 24 h. Then the solution of the carrier are separated, for example by decantation from the vessel, the nose ^{IR, p} rom ^y pu ^f rem. preferably containing a detergent (e.g., PBS pH 7.2 with ^0.05% by weight ^{and poly} ethylensc ox ^y = ^Rb tanmonolaurátu).

Determination of the complex proteolytic enzyme-mhíbitor Protek is prov-I by the carrier which has been coated with a specific antibody, specified enzyme or antigen binding fragments of antibodies, incubated · period in the range of one minute to ⁴ 8 hours n preferably after ^d OBU ² hours, a complex of proteolytic · ^Enzyme-y mhibitor proteases contained therein, k ^{p alinё,} such as the serum sample. In a parallel assay, a sample of known concentration of a proteolytic enzyme-protease inhibitor complex is used. These samples are prepared by diluting the reaction mixture of highly pure · protease highly pure protease inhibitor buffer ^{(staf-PBS, pH 7,} 2) or in HDS ^ ^p lazmě ^which was pre inaktvvována for 30 minutes at 56 ° C.

Thereafter, the treated carrier is freed from solution after incubation and washed to remove excess liquid and sample residues.

It is a buffer (e.g. PBS at pH 7.2). The carrier is then incubated with a solution of the labeled second reactive member. which is specific for a complex bound protease inhibitor. The incubation time depends on the particular .rozsa ^h at podmrne ^to with respect to ^P H while the temperature and concentration. Proceed with ^b equ .se time působert ^{p hy} the trend is in the range 2-24 ^s hodm. Redctanty for this step are reaMtí protdátky or antibody fragments which are yarn n ^yp roti antigennta determinants com ^p LEXN moleкЫ inhibitor bound proteolytic enzymes ^y tiokýc ^h.

Specific antibody preparations are isolated from the serum of specifically immunized animals. I ^to a suitably ^é turned obotacován antibody fractions .z. these antisera immunoadsorption procedure on immobilized antigen and separated by ^dp & ERN rotiláte ^{é unknown} specificity.

Značkovcí as materials for the second reactant is a ^beta ^^ i ^ ^í VaJ napřfalad enzymes. lummiscenčn ^s .značkovarn substance. Huorescent or radioactive substance. Within the scope of the present invention it is preferred to use such labeling enzymes. the activity of which can be readily determined by means of an optical test. especially when ^b arevné reactivity visible. worlds pn or ^change them, the concentration of NADH or NADPH ·. Typica ^Ké pNklady .značkovacích suitable enzymes are alkaline fofatázy. peroxidases. glucosooxidases ^γ . alpha- aiaktosid ^{g and} beyond. beta-galactosidase. . glucoamylase. invertase. maltdetydrogeaáza or. gtokoso-O-phosphateMehydrogenase.

Kov.alentm relation ^y značkovamch enz ^y m ^of the antibodies inhibitors of proteolytic speccfickými enz ^y m ^s ^ vázajfc anttyen or antibody fragments is ^also according .získaj from ^p Úsobí described in the literature [for example, J. Hlstochem. C ^y tochem. 22, 1084 (1974); Bull. Soc. Chim. Blol. 50, 1169 (1968)].

^{In the} test, only the ^I v'an ^s opttmálrt zředěrá značenýc ^{h and} that of the antibodies bound ^{and kk} omplexně nhibitorů proteolytlckých enzymes is detected using ^I prefilling ^Ez NYC ^h experiments must on ^no carrier to supply any unspecific compounds. i.e. that the presence of these compounds must not be detected on the carrier of the proteolytic enzyme-protease inhibitor complex. This К ^ú .se face ^to mites in ^the značltované antibody adding a detergent agent. for example, one millimole of sodium dodecyl sulfate.

After incubation, the solution is separated from the nose. napřftlad Shti ^Zn into ^BKY. carrier ^{for some} Koli washed monolaurate u ^{p f} rem. suitably containing detergens and the conventional detergent způobsahuje ^g ent, and then b together with the general from ^{of p} Úsobí determined amount of marker substance. ^and inflammation in ^é supported. To control ^for unspecific binding é marker substance on the carrier is carried ^out by following ^the same US j and ^k of the above described ^e. and ^{the width} and without ^the omplex · proteolytic enzyme protease inhibitor specified composition.

A complex of a proteolytic enzyme ·-in hlbitor protease to be determined ^{y y p} roteol tlckým enzyme in the biological sample are determined ^whose comparison with the above ^{p p} o Sanyo standard tests.

Come Prostřetek predtyženého ^{characterized} nátezu may additionally contain conventional stabilization čintála. j and ^k are the protetaová additives. (bovine serum aibumin or oval ^b umln). carbohydrates. glycerine. bacteriostatics. fungicides and the like.

The invention is illustrated by the following examples.

Example 1

Quantitative immunological evidence of leukocyte elastase and alpha-antirypsin complexes

(a) Manufacture of leukocyte elastase antisera

Elastase .se ZFE ^{Ka plated} Hdských polymorphic teu ^ cytů and that ^for long was purified by affinity chromatography and ion exchange chromatography c ^{h ICI.} to electrophoresis on elu ^{g p} ol ^y akrylamďdu at pH 4.3 and at ^f immunoelectrophoresis cropping with antisera human serum proteins are zjtetftelné ^Appl n ^s u ^ ^^ reaper other proteins. The lyophilized protein free soh is dissolved in 0.15 M sodium chloride solution to ^a concentration of ² m ^g / mh. One ml of this solution is emulsified with 1 ml of complete Freund's. adjuvant. This emulsion was injected sheep or goats until three ^hours čtyřec mfetech zadnfto runs in ^H and ^K u intramuscularly. The same injections are given even more ^of twice obdoW tower ^always three weeks, 21 days) · the last injection, blood is collected. He ^took the rnjetoe pofóvaj in point ^p in ¹² th nu ^d posledrnm after sampling ^to tearing. DIST ^{y 14} day ^p o n ⁱ c ⁱ is ^the odeMrá ^to rev. Serum from the blood of ^{ISK, and without} Nym from ^para uso ^b em. stabilizes and PRH ^d em ^{to 0th} ° 5% by weight sodium azide, and ^{the 0th 05%} by weight of acrylate natriumethylrtuťthiosalic ^y and stored at -20 ° C.

b] Production of alpha-antitrypsin antiserum

As an antigen using highly pure alpha-antůitypsm of ^L ^ ds ^{y a} l plasma. Antiserum alpha antitr kidding ^y is obtained from sheep or goats in the same manner. as described in paragraph (a) above. For the immunization of rabbits with 1 mg dissolved lyofii io ^ <v ^ <^ i ^ t ^ l ^ C ^ ^'alpha-beta antitrypsrnu Rosteh .soh in 0.5 ml of 0.1 ⁵ M sodium chloride solution. This solution is emulsified with ⁰ . ⁵ ml of complete Freund's adjuvant and the emulsion is injected intramuscularly to animals in three places at the rear ^run. Tejn ^{S p and} the emulsion was administered four weeks subltutánně to the neck of animals. After seven days, ^and the ^d © takes about ⁴⁰ ml of blood from the marginal ear vein. After four weeks, administered subcutaneously injected each other and at a distance ^of n ^d 7 .se ^ZDY discharged ^to the rev. Sé10 rum is treated as described in (a) above.

c) Isolation of imimoglobulin from the antisera The immunoglobulin fraction is isolated from the sera of the elastase or alpha-antitrypsin from the sera by conventional methods [Srand. J. Immunol. 2, Suppl. 1, 161 (1973) or Arch. Biochem. Biophys. 134, 279 (1969)] and stored in lyophilized salt-free form.

d) Purification of alpha-antitrypsin antibodies by immunoadsorption

The proteolytic enzyme inhibitor antibody is immunospecifically isolated by chromatography of purified imunglobulin (obtained according to paragraph c) J from an alpha-antitrypsin antiserum [according to (b)] via Senharose 4B or via a substituted agarose containing covalently bound alpha-antitrypsin. The proteolytic enzyme inhibitor antibody remains on the immobolized antigen and is eluted with 0.1 M glycine / hydrochloric acid buffer pH 2.8 or 3 M potassium rhodanide solution. In this way, about 5 to 20 percent by weight of the immunogtobulin present is immunospecifically isolated. Thus, immunoglobulin with a different specificity is removed in this way. The specific antibody eluate is dialyzed against phosphate buffered saline and then against 0.01 M ammonium bicarbonate solution. The protein obtained is then dried by lyophilization.

e) Production of enzyme-antibody conjugates

The immunospecificly purified antibody [according to d)] is cross-linked with an alkaline phosphatease from calf; small intestine using 0.2% by weight glutardialdehyde [Biochim. Biophys. Acta 251,427 (1971)]. The reaction product is chromatographed over a BioGel A column of 1.5 m (26 mm x 900 mm). Enzyme-antibody complexes were obtained in the first peak of AP-activity of the eluate. The solution obtained is stabilized by the addition of ovalbumin (0.1% by weight) and sodium azide (0.05% by weight) and stored at 4 ° C.

f) Coating the reaction vessels with elastase antibodies

The elastase antibodies produced by the method described in (a) and purified as described in (c) are immobilized by adsorption to the reaction plastic containers. Polystyrene tubes of 12 x 55 mm, round cuvettes or microtiter plates are used as reaction vessels. Typically, tubes or cuvettes are filled with 1 ml of 10 µg immunoglobulin / ml immunogloibulin solution (in 0.15 M sodium chloride solution with 0.02% sodium azide). For plates, 0.2 ml of a 50 µg / ml immunoglobulin solution is used per stock. The vials were sealed and then allowed to stand overnight at 4 ° C. Decant or aspirate liquid prior to use in the assay. The vials were then washed several times with a wash solution (0.15 M sodium chloride solution with 0.05% detergent and 0.02% sodium azide) and air-dried briefly.

g) Production of leukocyte elastase-alpha-antitrypsin complex

As an analytical sample for quantitative evidence, a complex is made from a proteolytic enzyme and a protease inhibitor in vitro by incubating both isolated, highly pure components. To this end, 0.2 ml of a solution of 0.5 mg of pure human leukocyte elastase in 1 ml [dissolved in 10 inM Tris / hydrochloric acid pH 7.4 buffer with 0.12 M sodium chloride] and 0.2 ml of the solution are mixed.

2.8 mg of pure alpha-antitrypsin (from human plasma) in 1 ml (dissolved in 50 mM Tris / hydrochloric acid buffer pH 8.8 with 0.05 M sodium chloride) and this mixture is then incubated for 2 hours at temperature 37 ° C. The mixture is then stored at -20 ^° C.

Analysis by immunoelectrophoresis showed that all elastase present in the incubation mixture had reacted to the elastase / .alpha.-antitrypsin complex.

h) Rediscovering a complex of leukocyte elastase and alpha-antitrypsin

For microtiter plates coated with the elastase antibody and prepared for use as described in (f), a 0.2 ml sample containing 0.1 to 10 ng of elastase in the depot inhibitor complex is filled. Samples with a defined elasitase content are obtained by diluting the enzyme / inhibitor mixture according to paragraph g); phosphate buffered saline is used as the diluent. To determine the comparative value, some of the depots are 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 depot are aspirated. It is then washed three times with phosphate-buffered saline and filled with the enzyme-antibody conjugate solution obtained in (e) (0.2 ml for depot). The solution had 800 units / L alkaline phosphatase activity. Dilution. the composition here was 10 mM Tris / hydrochloric acid 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. Air access to the platelets was blocked and incubated at room temperature overnight. After this incubation, the liquid is aspirated. The plates were washed three times and briefly air dried. Then, to determine the alkaline phosphatase activity, it is filled with a 0.2 ml buffer-substrate solution for the depot over a time series. After 10 minutes of incubation at room temperature, the reaction mixture from the depot is transferred to a microvoid and extinction is measured. The extinction value of the buffer solution and the substrate serves as zero. The protein-free depot represents extinction, which is a measure of the non-specific adsorption of the enzyme-antibody conjugate to the depot. An increased extinction was found in a depot containing proteolytic enzyme and protease inhibitor complexes. For evaluation, the observed extinction (minus extinction for non-specific adsorption) of the amount of elastase (ng) used in the inhibitor complex is compared. The following table gives the final extinction values at 405 nm after a 10 minute reaction time depending on the amount of elastase.

elastase (ng) OD405 / IC 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 - counter 0.0

The results show that the concentration of elastase-alpha-antitrypsin complexes can be quantitatively determined in this way. There is a close correlation between about 0.4 ng to 6 ng of complex bound elastase in the sample and the measured extinction. A buffer-substrate solution having the following concentrations in the assay was used: 10 mM p-nitrophenyl phosphate, 0.5 mM magnesium chloride, 1.0 mM diethanolamine / hydrochloric acid buffer pH 9.8.

Example 2

Quantitative immunological evidence of thrombin-antithrombin III complex

a) Isolation of immunoglobulin from antisera

Commercially obtainable rabbit antisera for human prothrombin and / or antithrombin IIII from human plasma are used as the source of specific protease antibodies and proteolytic enzyme inhibitors. Immunoglobulin fractions from these antisera are isolated by known methods and stored in lyophilized salt-free form.

b) Production of enzyme-labeled human anti-thrombin III antibodies

By the method of (a), the isolated immunoglohulin fraction from the antithrombin III antiserum of a human-labeled enzyme chemically covalently binds the calf small intestine alkaline phosphatase, and the immunoglobulin-enzyme conjugates produced in this way are further processed analogously as described in 1e).

c) Coating the reaction vials with prothrombin antibodies

According to paragraph a), the purified immunoglobulin fraction from the human prothrombin antiserum is immobilized by adsorption on plastic containers. To this end, polystyrene microtiter plates are used which are filled with 0.2 ml for a depot of a solution of 50 µg of immunoglobulin per ml (in 0.15 M sodium chloride solution with 0.02% sodium azide). The plates were airtight and allowed to stand overnight at 4 ° C. The liquid containing the immunoglobulin is removed prior to use in the assay. The vials were washed several times with 0.15 M sodium chloride solution, which still contained 0.05% detergent and 0.02% sodium azide, and then briefly air dried.

d) Production of molecular complexes from human thrombin and human antithrombin III

Human thrombin and antithrombin complexes for use as an analytical sample in the assay are prepared by incubating mixtures of both components in highly pure form. To this end, 50 units of antithrombin III (= heparin cofactor) and 50 units of heparin are incubated in a volume of 1.26 ml in 10 mM Tris / hydrochloric acid buffer pH 7.5 for 30 minutes at 37 ° C. After this incubation, 50 units of thrombin (corresponding to 16.7 g of enzyme protein) in 50 μΐ of the same buffer are added to the reaction mixture. After incubation for 2 hours at 37 ° C, the reaction solution and the same diluent are introduced into the assay.

e) Quantitative evidence of thrombin-antithrombin complexes

0.2 ml of a sample of known thrombin content in a complex bound form is filled into microtitre plate depots coated with prothrombin antibodies as described in (c). Phosphate buffered incubation solutions obtained in step (d) with graduated dilutions are used as samples of known content. To determine zero, some depots with 0.2 ml of diluent without enzyme-inhibitor complex were used. After incubating at room temperature for two hours, the contents of all the depots are aspirated and washed three times with phosphate buffered saline, which additionally contains 0.05% by weight of detergent. Then, the depots are filled with 0.2 ml of a solution of enzyme-210000 mem labeled antithrombin III antibodies produced according to b). The solution is preset to about 300 units / l. AP-activity. The diluent used was 10 mM pu-frutris / hydrochloric acid, pH 7.5, additionally containing 2 mM magnesium chloride, 0.025 mM zinc chloride, 1% ovalbumin, 1 mM sodium dodecyl sulfate and 0.02% sodium azide. . The plates are then airtight and incubated overnight at room temperature. After incubation, the liquid is aspirated, the plates are washed three times and air-dried briefly. Thereafter, 0.2 ml of buffer-substrate solution for the AP-test is filled with the depots. After an incubation time of 20 minutes, the reaction mixture from the depots is transferred to a microquay and the extinction is measured on a photometer. The extinction value of the buffer-substrate solution serves as the zero value.

Increased extinction values were found in depots containing proteolytic enzyme complexes with a protease inhibitor. For evaluation, extinctions at 405 nm of buffer-substrate solution from each depot were used after a 20 minute incubation compared to the calculated amount used in the bound thrombin complex.

The following table shows the final extinction values at 405 nm after a 20 minute incubation depending on the amount of antigen.

TABLE thrombin (ng)

OD405 / 20 minutes

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 - blind attempt 0

The results shown in the table show that there is a close association between the amount of antigen used and the photometric measured enzymatic indicator reaction, which allows quantitative determination of thrombin and antithrombin III complexes.

Claims (4)

1. A method of immunoassaying enzymes using an enzyme of interest, bound to a water-insoluble carrier, characterized in that the enzymes that form the enzyme-inhibitor complex with the inhibitor are incubated with the bound antibodies, the reaction solution is removed, the washed carrier with the antibody, saturated with the enzyme-inhibitor complex, is allowed to incubate with the coupling product formed from the antibody specifically reacting with the inhibitor and the labeled substance, the unbound coupling product is removed and the rate of the determined enzyme concentration is determined based on the concentration of labeled substance on the solid phase. 2. A composition for carrying out the method according to claims 1 to 3 comprising a reaction vessel coated with an antibody of the enzyme of interest and a coupling product consisting of an antibody specifically reacting with the inhibitor and a labeling substance. 3. A composition according to claim 2 comprising a reaction vessel coated with a proteolytic enzyme antibody and a coupling product consisting of an antibody specifically reacting with a protease inhibitor and a labeling substance. 4. The composition of claims 2 and 3 comprising a reaction vessel coated with a leukocyte elastase antibody and a coupling product consisting of an antibody specifically reacting with alpha-antitrypsin and a labeling enzyme.