EP0396692A1 - Procede d'analyse d'une enzyme, trousse et substances adaptes a ce prodede - Google Patents

Procede d'analyse d'une enzyme, trousse et substances adaptes a ce prodede

Info

Publication number
EP0396692A1
EP0396692A1 EP19890912141 EP89912141A EP0396692A1 EP 0396692 A1 EP0396692 A1 EP 0396692A1 EP 19890912141 EP19890912141 EP 19890912141 EP 89912141 A EP89912141 A EP 89912141A EP 0396692 A1 EP0396692 A1 EP 0396692A1
Authority
EP
European Patent Office
Prior art keywords
enzyme
inhibitor
sample
active free
active
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19890912141
Other languages
German (de)
English (en)
Inventor
Johan Hendrikus Verheijen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO
Original Assignee
Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO filed Critical Nederlandse Organisatie voor Toegepast Natuurwetenschappelijk Onderzoek TNO
Publication of EP0396692A1 publication Critical patent/EP0396692A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/972—Plasminogen activators
    • G01N2333/9726—Tissue plasminogen activator

Definitions

  • 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.
  • 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.
  • inactive proenzyme active enzyme
  • enzyme-inhibitor complexes enzyme-inhibitor complexes
  • inactive enzyme enzyme-inhibitor complexes
  • 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.
  • 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.
  • t-PA seems to play a role in fibrinolysis in vivo, probably because of its specific affinity for fibrin.
  • t-PA 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.
  • t-PA assays Two different types may be distinguished: immunological and functional assays.
  • 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.
  • 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.
  • 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 pr ⁇ teolytic 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.
  • 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
  • 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.
  • proteinaceous protease inhibitors for instance, are also unsuitable for use as inhibitor according to the invention.
  • 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.
  • 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).
  • 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.
  • 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.
  • aspartic acid proteases inhibitors have been described, often based on modified peptides such as statin or pepstatin.
  • inhibitors are known, based on aldehyde or ketone derivatives of amino acids or peptides, or based on phosphorus containing peptide analogs.
  • one or more groups are present which increase or determine the specificity for the target enzyme.
  • short peptides can be very suitable.
  • PPACK D-Phe-Pro-Arg-Chloromethyl ketone
  • u-PA urokinase
  • D-Glu-Gly- Arg-Chloromethyl ketone is a very good inhibitor of u-PA while t-PA reacts slowly with it.
  • a good specificity can be achieved for many enzymes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • samples which contain t-PA labelled with biotinyl-PPACK.
  • 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.
  • an enzyme such as horse radish peroxidase or alkaline phosphatase
  • 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.
  • a suitable substrate for . . horse radish peroxidase or alkaline phosphatase
  • 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.
  • 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.
  • 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.
  • the assay can be made suitable for other proteases.
  • 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.
  • 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 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
  • 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
  • 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.
  • 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.
  • 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.
  • a stabilizer such as mannitol
  • 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.
  • an anti-clotting agent such as sodium citrate
  • biotinyl-phe-pro-arg-chloro- methyl ketone a measured quantity of biotinyl-phe-pro-arg-chloro- methyl ketone.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé d'analyse d'une forme d'enzymes, notamment de la forme active libre d'une protéase telle que t-PA ou u-PA, consistant à prendre un échantillon, à lui ajouter directement un inhibiteur non macromoléculaire doté d'un groupe détectable, et ensuite à déterminer la quantité de l'enzyme active liée de cette manière. L'inhibiteur est de préférence une cétone d'halométhyle de peptidyle, et le groupe détectable est de préférence la biotine. L'invention comprend des inhibiteurs non macromoléculaires utiles dotés d'un groupe détectable, ainsi que des kits d'analyse comportant lesdits inhibiteurs non macromoléculaires dotés d'un groupe détectable.
EP19890912141 1988-11-04 1989-11-03 Procede d'analyse d'une enzyme, trousse et substances adaptes a ce prodede Withdrawn EP0396692A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL8802710 1988-11-04
NL8802710A NL8802710A (nl) 1988-11-04 1988-11-04 Werkwijze voor het bepalen van een enzym en daarvoor geschikte kit en stof.

Publications (1)

Publication Number Publication Date
EP0396692A1 true EP0396692A1 (fr) 1990-11-14

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ID=19853169

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19890912141 Withdrawn EP0396692A1 (fr) 1988-11-04 1989-11-03 Procede d'analyse d'une enzyme, trousse et substances adaptes a ce prodede

Country Status (4)

Country Link
EP (1) EP0396692A1 (fr)
JP (1) JPH03503486A (fr)
NL (1) NL8802710A (fr)
WO (1) WO1990005309A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9340820B2 (en) 2009-08-26 2016-05-17 Queen's University Of Belfast Compounds and methods for protease detection

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL115899A (en) * 1994-11-17 2002-07-25 Basf Aktiengesellshaft History of Acid-2 [(2-alkoxy-6-trifluoromethyl-pyrimidine-4-yl) oxymethylene] -phenylate, their preparation, preparations for the control of animal and fungal pests containing these histories and a number of their intermediates
EP0759556A3 (fr) * 1995-07-24 1998-05-20 BEHRINGWERKE Aktiengesellschaft Méthode pour la quantification des facteurs actifs
AU1801101A (en) * 1999-11-22 2001-06-04 American Red Cross Novel detection method for a functionally active form of an enzyme in biologicalsamples and a kit
AU2002367022A1 (en) * 2001-12-21 2003-07-30 Immunochemistry Technologies, Llc Novel affinity labels
JP7355140B2 (ja) * 2022-02-28 2023-10-03 住友ベークライト株式会社 セリンプロテアーゼの検出用または測定用試薬

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4318904A (en) * 1980-04-25 1982-03-09 Research Corporation Peptide affinity labels for thrombin and other trypsin-like proteases
US4438209A (en) * 1981-07-17 1984-03-20 Mallinckrodt, Inc. Radioimmunoassay for fibrinopeptide A
JPS58129998A (ja) * 1981-11-02 1983-08-03 ジエ−ムス・ウオルタ−・リヤン 標識蛋白質性阻害剤を用いるプロテア−ゼの分析法
NL8201987A (nl) * 1982-05-13 1983-12-01 Tno Werkwijze voor het bepalen van de activiteit van plasminogeenactivator van het weefseltype, alsmede voor gebruik bij deze werkwijze geschikte combinatie van het "kit"-type.
DE3512909A1 (de) * 1985-04-11 1986-10-23 Behringwerke Ag, 3550 Marburg Verfahren zur bestimmung von plasminogenaktivatoren (pa)
CA1313488C (fr) * 1986-03-12 1993-02-09 Adair John Hotchkiss Dosage

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9005309A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9340820B2 (en) 2009-08-26 2016-05-17 Queen's University Of Belfast Compounds and methods for protease detection

Also Published As

Publication number Publication date
WO1990005309A1 (fr) 1990-05-17
NL8802710A (nl) 1990-06-01
JPH03503486A (ja) 1991-08-08

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