EP0396692A1 - A method of assaying an enzyme and kit and substances suitable for that method - Google Patents

Abstract

The invention relates to a method for analyzing a form of enzymes, in particular the free active form of a protease such as t-PA or u-PA, consisting in taking a sample, in directly adding a non-inhibitor to it. macromolecular with a detectable group, and then determining the amount of the active enzyme linked in this way. The inhibitor is preferably a peptidyl halomethyl ketone, and the detectable group is preferably biotin. The invention includes useful non-macromolecular inhibitors with a detectable group, as well as analysis kits comprising said non-macromolecular inhibitors with a detectable group.

Description

A method of assaying an enzyme and kit and substances suitable for that method.

The invention relates to a method of assaying the concentration of a form of an enzyme in a biological fluid which also contains inhibitors that react with the enzyme, by isolating a sample from the fluid and assaying the concentration of the form of that enzyme in such sample by means of an immunological assay method.

In biological fluids, such as tissue extracts, urine, cell extracts, culture media, plant extracts or blood, many types of enzymes occur. In these fluids the enzymes are often not very stable, for instance because of the presence of other enzymes or physiological inhibitors, or because the enzyme is intrinsically unstable. In many instances such enzymes occur in low concentrations and therefore it is not easy to assay them accurately. In blood, for instance, proteases occur, which are involved in coagulation, fibrinolysis, immune reactions, and histogenesis or degradation processes. Examples of such proteases are thrombin, plasminogen activators, plasminogen, elastase, and collagenase. These proteases are often present in different forms, viz. as inactive proenzyme, active enzyme, enzyme-inhibitor complexes and inactive enzyme. Mostly the interest is in the free active enzyme, but sometimes also or especially in the enzyme present as proenzyme or inhibitor complex. Assaying this form of the enzyme is often difficult, for instance because the active free enzyme may still react with inhibitors occurring naturally in the sample (physiological inhibitors) after the sample has been taken, so that the real value in the sample will be higher than the value measured in the test.

This problem may prove quite a hindrance. for instance in assaying the plasminogen activators t-PA (tissue-type plasminogen activator) and u-PA (urokinase) in blood, because these enzymes occur in very low concentrations and blood contains a very fast physiological inhibitor. The same problem presents itself with a number of clotting enzymes, such as thrombin.

Plasminogen activators are very specific serine proteases which catalyze the conversion of plasminogen to plasmin. Plasmin is capable of dissolving blood clots by breaking down the fibrin present in them. Various plasminogen activators are known, of which urokinase and tissue-type plasminogen activator are the most important. In particular, t-PA seems to play a role in fibrinolysis in vivo, probably because of its specific affinity for fibrin. In this connection there is a need for a reliable and simple method of determining the concentration of active t-PA in body fluids, in particular blood, of both healthy people and patients having certain disorders, for instance thrombosis occurring at an early age or unexplained bleeding. In body fluids t-PA, like other proteases, may occur in different forms, namely as active free enzyme, as inactive free enzyme, and as an inactive complex with inhibitors that occur physiologically. For in-vivo fibrinolysis probably only the active free t-PA is important.

Two different types of t-PA assays may be distinguished: immunological and functional assays. in immunological assays the amount of plasminogen activator antigen is determined. Depending on the antibody used either total t-PA antigen is determined, or one or more of the t-PA forms: active free t-PA, inactive free t-PA, or inactive t-PA in complex with inhibitors. In functional assays only the active free t-PA is determined. Of both types of assay methods a number of examples are known. However, in these assays determining the active free t-PA in body fluids such as blood remains a problem. The point is it has been found that such fluids often contain inhibitors which are capable of reacting after the blood has been drawn and thus lower the amount of active free t-PA, as a result of which an incorrect value will be measured. In the clinical practice it has proved hard to effect a rapid standardized blood-collecting procedure in order to overcome these problems.

In European patent application EP-A-0 237 332 it is proposed in a method of determining the plasma concentration of t-PA and other coagulation and fibrinolysis components to inhibit the prόteolytic activity of t-PA directly after the blood sample has been taken by adding an appropriate inhibitor, in particular D-Phe-Pro-Arg-chloromethyl ketone. Later, then, a suitable immunoassay follows, in which the total of t-PA antigen present is determined. However, that method can only be used to determine the total amount of t-PA present and cannot give any information on the amount of active free t-PA present, unless that amount is very much larger than that of all other forms of t-PA together. This last situation occurs only in the case of an infusion of pharmacological quantities of t-PA as a thrombolytic agent, so that the practical use of the method according to EP-A-0 237 332 would seem to be limited to monitoring the effects of thrombolytic therapy with t-PA, which monitoring is the object of the European application mentioned above. Under normal conditions, i.e. outside the situation involving thrombolytic therapy with t-PA, the amount of free active t-PA in blood is mostly very much smaller than the amounts of inactive and/or inhibitor-bound t-PA. In those cases the procedure described in European patent application EP-A-0 237 332 cannot be used to measure free, active t-PA.

The procedure described in European patent application EP-A-0 070 478, in which proteolytic enzymes present in a blood sample are inhibited or deactivated

5 by adding rapidly acting protease inhibitors, is also unsuitable for assaying these proteolytic enzymes themselves. This method can only be used to assay the substrates of the enzymes present in the sample, for instance fibrinopeptide A.

10 It is an object of the present invention to increase the reliability of methods of determining the concentration of an active free enzyme, as in particular the proteases t-PA and u-PA, in biological fluids, such as blood, and culture media, containing

15 in addition one or more physiological inhibitors of the enzyme, and in particular to lessen the dependency on the lapse of time between the moment when the sample is taken and the time of the assay.

This object is realized, according to the

20 present invention, by providing a method of the type defined in the preamble, which is characterized in that when the form of the enzyme to be assayed is in the free state, during or virtually directly after the taking of the sample, and when the form to be

25. assayed is a proenzyme or an enzyme inhibitor complex, at the latestvirtually directly after its activation, a sufficient amount of a non-macromolecular inhibitor of the enzyme provided with a detectable group is introduced into the sample to bind substantially all

30 active free enzyme present before it can react with the physiological inhibitors present in the sample, and at a later time the amount of the active free enzyme bound in this way is determined by means of an immunological assay. According to the present invention

35 the sample is directly incubated with an excess of inhibitor of the enzyme concerned if the form to be assayed is the active enzyme in the free state. This inhibitor contains at least two reactive groups, one of which reacts with a certain specificity with the active centre of the enzyme and one of which either contains a label that can be specifically demonstrated, such as a radioisotope, or is otherwise detectable, for instance by means of a specific interaction with a reagent that contains a detectable label. Because only the free and active enzyme will react with the inhibitor, only that enzyme will be labelled and later on be assayed. A fast reaction with the active free enzyme in the sample prevents a reaction with physiological inhibitors or other inactivation after the sample has been taken or during storage. The inhibitor is a non-macromolecular inhibitor selective for the enzyme to be assayed. Macromolecular substances, such as antibodies which react with the active centre of the enzyme to be assayed, are less suitable for use as inhibitors according to the invention. Reasons for this are 1) that the reaction rate of a free enzyme with a large molecule such as an antibody is low, as a result of which a high concentration of the antibody would be necessary; 2) that antibodies are not very stable during transport and storage; 3) that the sensitivity of the assay is rather low; and 4) that an antibody- antigen reaction is reversible so that the reaction with physiological inhibitors remains possible, in particular when those inhibitors have an irreversible activity. For these reasons naturally-occurring proteinaceous protease inhibitors, for instance, are also unsuitable for use as inhibitor according to the invention. For that matter, the use of such proteinaceous natural inhibitors is described in European patent application EP-A-0 080 279 in respect of a method of quantitating proteases, which comprises using the proteinaceous inhibitor in a labelled form and measuring the amount of label bound to the protease after the reaction. However, the reaction rate of the proteinaceous inhibitors used is too low for these inhibitors to be able to compete adequately with, for instance, the protease inhibitor, which occurs naturally in blood, (the plasminogen . activator inhibitor). When blood samples are treated according to the method described in EP-A-0 080 279, therefore, only a part of the free active t-PA will be labelled, and so the result of the assay will turn out too low. Moreover, the magnitude of the error varies and depends on the strongly fluctuating amounts of plasminogen activator inhibitor that may be present in the sample. In the present invention these problems are obviated by using low-molecular synthetic inhibitors, which react very rapidly with active free enzymes, such as the proteases specifically used herein to exemplify such enzymes, are present in high molar concentrations and are stable during transport and storage. It is important that these inhibitors have a high affinity for the target enzyme, react rapidly, in any case much more rapidly than the competing physio¬ logical inhibitors, preferably enter into a covalent bond with the enzyme, are stable during storage and transport, and preferably show a high degree of specificity for the target enzyme. Many of the enzymes occurring in biological fluids belong to the group of proteases. Different types of proteases occur, such as serine proteases, thiol proteases, aspartic acid proteases, and metalloproteases. The clotting and fibriolytic (pro)enzymes occurring in blood such as thrombin, plasminogen, tissue-type plasminogen activator (t-PA) and urokinase (u-PA), are serine proteases. An important thiol protease is cathepsin B from the lysosomes. The stomach enzyme pepsin belongs to the group of aspartic acid proteases, while collagenase, involved, among other things, in bone metabolism is a metalloprotease.

For many enzymes in each of these groups inhibitors are known, often having high affinity and high specificity. Thus serine proteases are for instance inhibited by organic fluorophosphates (as diisopropylfluorophosphate),. sulphonyl fluorides (as phenylmethane sulphonyl fluoride) and peptidylhalomethyl ketones. Thioproteases can also be inhibited by peptidylhalomethyl ketones, pepti- dyldiazomethanes or peptidylaldehydes. For various aspartic acid proteases inhibitors have been described, often based on modified peptides such as statin or pepstatin. For metalloproteases, too, inhibitors are known, based on aldehyde or ketone derivatives of amino acids or peptides, or based on phosphorus containing peptide analogs. In general, in addition to a reactive group which reacts with the active centre, one or more groups are present which increase or determine the specificity for the target enzyme. For this purpose short peptides can be very suitable. Thus it is known that D-Phe-Pro-Arg-Chloromethyl ketone (PPACK) is an excellent inhibitor of thrombin and t-PA, while urokinase (u-PA), another plasminogen activator that occurs in blood, reacts rather slowly with it. D-Glu-Gly- Arg-Chloromethyl ketone, on the other hand, is a very good inhibitor of u-PA while t-PA reacts slowly with it. By means of a suitable combination of a reactive group and peptide, a good specificity can be achieved for many enzymes. In the present invention derivatives of such synthetic inhibitors are described that in addition to the functional groups necessary for inhibiting and specifically recognizing the enzyme, contain a label or a group that is recognizable by other means, which can later be detected in a simple and sensitive way. In this respect the invention is essentially different from the methods according to EP-A-0 237 332 and EP-A-0 070 478, in which unlabelled inhibitors are used. Different types of labels or recognizable groups can be used, such as radioactive isotopes, fluorescent groups (for instance a dansyl group), haptens detectable by means of antibodies (for instance trinitrobenzene), biotin (derivatives) detectable by means of (strept)avidin, enzymes that provide a detectable product, or other groups that are detectable or can be made detectable by means of physical, chemical, biological, or immunological methods.

Accordingly, when the sample is contacted with an inhibitor derivative of the type mentioned directly after or even during sampling, the free protease present, as t-PA, will react immediately and irreversibly with this derivative. Thus, not only is the reaction with the physiological inhibitors present in the sample prevented, but the free t-PA is provided with a detectable label.

As a result the amount of free t-PA which was present at the time of the sampling can later be distinguished from the t-PA that is present in different forms. The procedure of sampling and storage is thus very much simplified. If a proenzyme or an inactive inhibitor complex is to be measured, a suitable activation step can be taken-before the inhibitor derivative is added, in order to convert the inactive proenzyme or inhibitor complex to an active enzyme. If the concentration of the proenzyme or the inhibitor complex is to be assayed excluding the concentration of the active free enzyme, for example, a separate assay of the active free enzyme can be carried out, the difference between the two measuring results providing information about the concentration sought, or the active free enzyme can be prevented from contributing to the measuring results, for example, by adding inhibitor without a recognizable group directly during sampling. The labelled inhibitor to be used according to the invention can readily be prepared according to conventional techniques. To this effect one starts for instance from commercially available inhibitor preparationswith a specificity suitable for the enzyme to be assayed. For t-PA one could think of D-Phe-Pro-Arg- chloromethyl ketone, while for u-PA Glu-Gly-Pro-Arg- chloromethyl ketone is more suitable. A detectable group is coupled to such an inhibitor, for instance to a free NH2 group, optionally via a spacer of the desired length. A suitable group for this purpose is for instance biotin which can be coupled as N-hydroxy- succinimidylbiotin to the free amino group of the above inhibitors for instance. Because, in aqueous solution, chloromethyl ketons have their highest stability at low pH and the coupling with N-hydroxysuccinimidyl group preferably occurs at high pH, it is advisable to conduct the coupling in an organic solvent. A suitable solvent for this purpose is methanol to which preferably an organic base such as triethylamine has been added to bind the protons released during the coupling reaction. The temperature at which the coupling reaction can be conducted is not critical but is preferably around room temperature. Under such conditions the coupling reaction will be completed after about 3 hours. The product can be purified, for instance by means of reversed phase HPLC using a gradient starting with 0.1% trifluoroacetic acid in water and ending with 0.1% trifluoroacetic acid in acetonitrile. The solvent can be removed by evaporation, preferably in vacuo. The product can be dissolved in an organic solvent or in water. Methanol is very suitable for the purpose.

In a concrete case the inhibitor D-Phe-Pro-Arg- chloromethyl ketone and the label biotin were used. Starting from D-Phe-Pro-Arg-chloromethyl ketone and

N-hydroxysuccinimidyl biotin the inhibitor derivative biotinyl-Phe-Pro-Arg-chloromethyl ketone was prepared by reacting D-Phe-Pro-Arg-chloromethyl ketone 5 mM with N-hydroxysuccinimidyl biotin 5 mM in methanol with triethylamine 10 mM for 0-24 h at room temperature. After different reaction times samples were taken and the conversion was monitored by means of HPLC using a reversed phase column and elution with a gradient from 0.1% trifluoroacetic acid in water ^"to 0.1% trifluoroaceti acid in acetonitrile. The peaks of the two starting materials proved to decrease clearly and a peak of the coupling product appeared. After about 3 h of incubation the conversion was maximal. The product was purified by means of preparative HPLC as outlined above. The biotinyl -PPACK is an excellent inhibitor of t-PA, comparable with PPACK itself.

To a t-PA containing sample, whose concentration of free t-PA is to be assayed, an excess of biotinyl- PPACK is added, preferably in a concentration of 0.1-1um to achieve a sufficiently rapid reaction of t-PA and inhibitor. Then follows an assay that strongly resembles a traditional enzyme immunoassay. Synthetic plastics containers are used, for instance polyvinyl chloride or polystyrene containers, preferably in the form of tubes or microtiter plates with a plurality of separate wells. These containers are coated with monoclonal or polyclonal antibodies against t-PA and then coated once again with for instance bovine serum albumin according to the conventional procedures for that purpose. To the coated containers samples are added which contain t-PA labelled with biotinyl-PPACK. After incubation with shaking for 1-24 h in a buffer which is suitable for^" the purpose, such as a 10 mM phosphate buffer having a pH of for instance 7.5, and preferably containing 5 mM ethylenediaminotetraacetate and a detergent such as Tween 20, 5 g/1, the containers are washed several times with the same or a similar buffer. Then streptavidin or avidin is added, coupled with an enzyme such as horse radish peroxidase or alkaline phosphatase, followed by incubation with shaking for 1-4 h at for instance room temperature. The containers are washed again with a suitable buffer and finally incubated with a suitable substrate for . . horse radish peroxidase or alkaline phosphatase, yielding a coloured product. As in enzyme immunoassays the concentration in a certain area is approximately propor- tionate to the amount of free t-PA in the original sample. The detection limit of the present method is such that assaying physiological concentrations of t-PA in blood or other body fluids is possible. The great advantage of the present method over and above existing procedures is that the situation existing in the sample at a certain moment, preferably directly after or during sampling, or at any other appropriate instant, for instance after activation of the proenzyme or inhibitor complex, can be fixed, whereafter the actual assay can be carried out at an appropriate time. For routine assays of large numbers of samples the assay is preferably carried out in microtiter plates and the measurements are conducted with special multichannel photometers, optionally linked to a computer for data storage and processing. However, the assay may also be carried out in tubes or cuvettes. The specificity of the present method is determined by two factors, viz. the choice of the inhibitor derivative and the choice of the polyclonal or monoclonal antibody in the detection.

By adapting the inhibitor derivative or the antibody or both, the assay can be made suitable for other proteases. Examples are (pro)enzymes occurring in clotting or fibrinolysis, such as plasminogen, u-PA, t-PA, (pro)thrombin, but the principle described here is generally applicable. Other examples of enzymes that could be assayed in a similar way are, in addition to the serine proteases, the thiol proteases such as cathepsin B for which derivatives of Phe-Phe-Arg- chloromethyl ketone can be used as inhibitors, aspartate proteases such as renin with derivatives of statin as inhibitors, metalloproteases as collagenase and angiotensin converting enzyme with for instance ketomethylene and aminoketone peptide derivatives.

The invention further provides a kit-type combination of agents for carrying out the assay according to the inve mntion, comprising

(a) an inhibitor of the active free enzyme to be assayed, which inhibitor carries a detactable group, and

(b) a polyclonal or monoclonal antibody against the active free enzyme to be assayed.

In addition to a container filled with (a) and a container filled with (b) such kits will as a rule comprise still other means and one or more instructions on how to use the kit. The other means referred to, include for instance a container filled with a buffer solution which optionally includes a preservative and a surface- active agent; when an inhibitor derivative labelled with a biotin (derivative) or hapten is used, a container with a measured quantity of (preferably enzyme-)labelled streptavidin or avidin, or an enzyme-coupled monoclonal or polyclonal antibody against the hapten; when a detection method based on an enzymatic reaction is used, containers with a substrate suitable for such an enzyme, for instance horse radish peroxidase or alkaline phosphatase, or containers filled with incubation fluid for carrying out the conversion of this substrate.

A combination according to the invention may also comprise a container with a measured quantity of the enzyme or proenzyme to be assayed, in order tomake control measurements possible. The invention finally comprises also inhibitors of an active free enzyme, carrying a detectable group, which are suitable for use in the method according to the invention.

The invention is illustrated in and by the following example. EXAMPLE

(a) Inhibiting t-PA A solution containing 5 IU/ml t-PA was incubated with 1 xiΑ of a purified biotinyl-PPACK preparation for some minutes at a temperature between 0° and 37°C, preferably at room temperature. Then the residual t-PA activity was measured by a known method to that effect. It turned out the t-PA activity was almost completely inhibited by the inhibitor preparation. This means that the inhibitor preparation is capable of forming a covalent complex with the active centre of t-PA.

(b) Assaying t-PA To a t-PA containing sample biotinyl-PPACK was added as an inhibitor preparation and incubation took place as under (a). A solution containing 10ug/ml of polyclonal antibodies which had been produced against t-PA (available from Biopool, Sweden) in 0.1 M NaHCθ3,.pH 9.6, was contacted with the wells of a PVC microtiter plate for about 20 h at room temperature and with shaking. A volume of 0.10 ml of such a solution per well was very suitable for the purpose. After-coating with for instance 10 mg/1 of bovine serum albumin in the same buffer for some hours proved to be highly recommendable.

Various amounts of a sample incubated with a biotinyl- PPACK inhibitor preparation were introduced into the wells of a microtiter plate coated as indicated above. The volume was adjusted to 0.10 ml per well with a buffer containing 10 mM of sodium phosphate, 0.15 M NaCl, 5 mM EDTA and 0.5 g/1 Tween 20. Overnight the plate was lightly shaken at room temperature. After the wells had been emptied they were washed three times with 0.15 ml of the same buffer. Next, 0.10 ml of a solution of a streptavidin-horse radish peroxidase conjugate in the same buffer was placed in each well. After incubation for 2 h at room temperature the plate was washed again three times with the same buffer. Then 0.10 ml of a solution of tetramethylbenzidine in a urea peroxide containing buffer was placed in each well. After incubation for 0-2 h at 25°C 0.05 ml of 2 M H2SO4 was added and the extinction was measured at 450 nm by means of a multi-channel photometer. It was found that the measured extingtion, increased virtually linearly with the amount of free t-PA in the sample, (c) Kit for t-PA assay A "kit" for 96 assays as described under (b) comprises: A microtiter plate coated and after-coated according to (b), provided with a stabilizer such as mannitol; a container with a sufficient amount (0.1G ,u mole) of biotinyl-Phe-Pro-Arg-chloromethyl ketone in dry form or dissolved in 1.0 ml methanol; a container with 0.50 ml tetramethylbenzidine in the liquid form; a container with a measured amount of urea peroxide, preferably in tablet form; optionally, a container with a known amount of purified t-PA in the dry form, to which water must be added, for carrying out control measurements; operating instructions according to (b) and instructions on how to prepare the samples and how to collect the blood. Instead of a storage container with inhibitor preparations, the kit may comprise 96 ready-for-use blood collecting tubes, for instance of the vacuum type, containing a measured quantity of an anti-clotting agent such as sodium citrate and a measured quantity of biotinyl-phe-pro-arg-chloro- methyl ketone.

Claims

1. A method of assaying the concentration of a form of an enzyme in a biological fluid which also contains inhibitors that react with the enzyme, by isolating a sample from the fluid and determining the content of the form of said enzyme in such sample by means of an immunological assay method, characterized in that when the form to be assayed is the active enzyme in the free state, during or virtually directly after the isolation of the sample, and when the form to be assayed is a proenzyme or an enzyme-inhibitor complex, at the latest virtually directly after its activation, a sufficient amount of a non-macromolecular inhibitor of the enzyme provided with a detectable group is introduced into the sample to bind substantially all active free enzyme present before it can react with the physiological inhibitors present in the sample, and at a later time the amount of the active free enzyme bound in this way is determined by means of an immunological assay.

2. A method according to claim 1, characterized in that the concentration of the active enzyme in the free state in the biological fluid is determined by adding the inhibitor provided with a detectable group during or virtually directly after the isolation of the sample and later the amount of active free enzyme bound to said inhibitor provided with a detectable group is determined by means of an immunological assay.

3. A method according to claim 1, characterized in that the enzyme is a protease.

4. A method according to claim 1, characterized in that the enzyme is a serine protease.

5. A method according to claim 4, characterized in that an organic fluorophosphate, and organic sulphonyl fluoride or a peptidyl halomethyl ketone is used as inhibitor.

6. A method according to claim 4, characterized in that to assay active free tissue-type plasminogen activator (t-PA) or activated t-PA/inhibitor complex, D-Phe-Pro-Arg-chloromethyl ketone is used as inhibitor.

7. A method according to claim 4, characterized in that to assay active free urokinase (u-PA) or activated pro-u-PA, D-Glu-Gly-Arg-chloromethyl ketone is used as inhibitor.

8. A method according to any one of claims

1-7, characterized in that an inhibitor is used that is provided with biotin as detectable group.

9. A method of assaying the concentration of active free tissue type plasminogen activator (t-PA) in blood, by isolating a sample from the blood and determining the active free t-PA content of such sample by means of an immunological assay method, characterized in that virtually directly after isolating the sample a sufficient amount of biotinyl-Phe-Pro-Arg-chloromethyl ketone is introduced into the sample to bind essentially all active free t-PA present before it can react with the physiological inhibitors present in the sample, and at a later time the amount of active free t-PA bound in this way is determined by means of an immuno- logical assay.

10. A kit-type combination of agents for ^• carrying out the method according to any one of the preceding claims, comprising

(a) a non-macromolecular inhibitor of the active free enzyme to be assayed, which inhibitor is provided with a detectable group; and

(b) a polyclonal or monoclonal antibody against the active free enzyme to be assayed.

11. A non-macromolecular inhibitor of an active free enzyme, provided with a detectable group and suitable for use in the method according to any one of claims 1-9.