WO1990004038A1 - Process for determining the activity of transglutaminases - Google Patents

Process for determining the activity of transglutaminases Download PDF

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Publication number
WO1990004038A1
WO1990004038A1 PCT/EP1989/001169 EP8901169W WO9004038A1 WO 1990004038 A1 WO1990004038 A1 WO 1990004038A1 EP 8901169 W EP8901169 W EP 8901169W WO 9004038 A1 WO9004038 A1 WO 9004038A1
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WIPO (PCT)
Prior art keywords
substrate
fibrinogen
activity
transglutaminase
solid phase
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PCT/EP1989/001169
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German (de)
French (fr)
Inventor
Hans-Rudolf KLÖSS
Gert MÜLLER-BERGHAUS
Eberhard Selmayr
Original Assignee
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO1990004038A1 publication Critical patent/WO1990004038A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/52—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving transaminase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
    • 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
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91074—Aminoacyltransferases (general) (2.3.2)
    • G01N2333/9108—Aminoacyltransferases (general) (2.3.2) with definite EC number (2.3.2.-)
    • 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
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91074—Aminoacyltransferases (general) (2.3.2)
    • G01N2333/9108—Aminoacyltransferases (general) (2.3.2) with definite EC number (2.3.2.-)
    • G01N2333/91085—Transglutaminases; Factor XIIIq (2.3.2.13)

Definitions

  • the invention relates to a method for determining the enzymatic activity of transglutaminases in plasma, cells and tissues by crosslinking specific labeled protein substrates and to methods for producing the labeled substrates.
  • the determination method according to the invention can be used to detect certain diseases, to monitor a course of the disease or to control a therapy.
  • Transglutaminases are the members of a family of enzymes which are known to covalently crosslink extracellularly and intracellularly molecules of different structural proteins by enzymatic formation of a £ - ( ⁇ - glutamyl) lysyl bond (reviews: a. Lorand L. and Conrad, SM, Transglutaminases, Molecular and Cellular Biochemistry 58: 9-35 (1984); b. Folk, JE and Finlayson, JS, The £ "" ( ⁇ - glutamyl) lysine crosslink and the catalytic role of transglutaminases, Advances in Protein Chemistry 31: 1-133 (1977).
  • the most important enzyme in the transaminase family is blood coagulation factor XIII.
  • the following are known as substrates: fibrin, fibronectin, alpha-plasmin inhibitor, thrombospondin, collagen, alpha 2 -macroglobulin, von Willebrand factor, factor V, actin and myosin.
  • Transglutaminases are involved in blood coagulation, thrombosis, regulation of fibrinolysis, consumption coagulopathy, wound healing, tumor metastasis, arteriosclerosis, congenital factor XIII deficiency, spontaneous abortion, rheumatism, inflammatory and immunological - Z -
  • the object of the invention is therefore to develop a reagent or method to specifically and quantitatively determine the enzymatic activity of transglutaminases and in particular factor XIII.
  • the test should be easy and quick to perform and should be able to record a large number of samples in parallel with great accuracy.
  • this object is achieved by a method for determining the activity of transglutami noses, which is characterized in that a solution of a physiological substrate of the transglutaminase to be determined, which carries a label, is incubated with a solid phase of the enzyme in the presence of the sample to be examined, the phases are separated and the Marking present in the solid or in the liquid phase is determined as a measure of the transglutaminase activity.
  • any physiological substrate of the transglutaminase of interest in each case can be used as the transglutinase substrate.
  • the substrates fibrin, fibronectin, alpha ⁇ plasmin inhibitor, thrombospondin, collagen, alpha 2 macroglobulin, von Willebrand factor, factor V, actin and myosin are suitable for determining the physiologically particularly important trans-aminase blood coagulation factor XIII .
  • Fibrin is particularly preferably used as an in situ substrate in the form of fibrinogen, from which fibrin is formed during the determination by the action of thrombin.
  • the same enzyme substrate is preferably used in the liquid as in the solid phase, although this is not essential to the invention and the only thing that matters is that the substrate of the enzyme to be determined is used as substrate at all.
  • the enzyme combines the two substrates, the labeled substrate present in the liquid phase and the substrate bound to the solid phase, in a type of crosslinking reaction. After separation of the phases, the labeled substrate bound to the solid phase by the crosslinking reaction can be determined on the basis of its marking. The amount of connected! marked substrate correlates the sought enzyme - H -
  • Radioactive isotopes such as e.g. Iodine isotopes, dyes, fluorescent dyes, enzymes, as well as the partners of physiological binding pairs such as e.g. Biotin / streptavidin or antigen / antibody.
  • the partners of physiological binding pairs such as e.g. Biotin / streptavidin or antigen / antibody.
  • biotin / fibrinogen is preferably used as the labeled soluble substrate in the context of factor XII determination and detected with a peroxidase-streptavidin conjugate using the usual peroxidase determination method.
  • the determination of peroxidase via color reactions is familiar to the person skilled in the art and does not require any further description here.
  • Biotin - ⁇ - aminocaproic acid-N-hydroxysuccinimide ester (BX-NHS) has proven to be particularly advantageous for labeling the dissolved substrates by means of biotin, since it maintains the biological activity of the sensitive substrates such as fibrinogen.
  • Fibrinogen is expediently used in a purified form for use as a substrate.
  • Mahn, I. and Müller-Berghaus, G. Studies on catabolism of 125-1-labeled fibrinogen in normal rabbits and in rabbits with indwelling intravenous catheters: Methodology aspects is particularly suitable for cleaning. Haemostasis 4_: 40-50 (1975)).
  • the marking bound to the solid phase by the crosslinking reaction is determined, it has proven to be advantageous to coat the solid phase with a buffered solution of a chaotropic substance, in particular with a urea solution or with dilute acid clean unbound soluble substrate.
  • Suitable substrates for the solid phase and carrier of the substrate present in the solid phase are those which are usually suitable for enzyme reactions, such as e.g. Carrier material based on carbohydrates such as agarose, cellulose, starch or the like or based on plastics as used in particular for microtiter plates.
  • the substrate can be bound to the solid support by adsorption, by forming a covalent bond with the substrate or by crosslinking the substrate on the support surface. Because of the simplicity of production and the direct applicability for the method according to the invention, adsorptive binding to microtiter plates is particularly suitable.
  • the carrier surface is advantageously treated or coated with a non-crosslinkable non-specific protein such as bovine serum albumin or milk protein.
  • Another object of the invention is a reagent for determining the activity of transglutaminases, which is characterized in that it contains a solid-phase-bound physiological transglutaminase substrate and a water-soluble labeled physiological transglutaminase substrate.
  • the solid-phase-bound substrate is expediently prepared using a solution which contains about 0.1 to 10 mg / l of substrate and is contacted with the solid support with binding of the substrate. With adsorptive binding to the carrier, this leads to an adequate loading of the carrier. With microtiter plates as supports and adsorptive binding, 0.5 to 5 ⁇ g substrate / well (well) gave good results.
  • the soluble labeled substrate is generally used in a concentration of 0.05 to 10 mg / ml, preferably 0.5 to 2 mg / ml.
  • a preferred reagent which is suitable for determining the activity of factor XIII, contains fibrinogen bound to a solid phase, labeled soluble fibrinogen and thrombin.
  • this reagent contains, as soluble substrate, biotin fibrinogen, and, before use, separately streptavidin-peroxidase conjugate and a detection system for peroxidase.
  • the invention enables a simple, rapid determination of the transglutaminase activity, which can be used advantageously for the diagnosis and therapy of diseases associated with the transglutaminases.
  • FIG. 1 shows a graphic representation of the adsorption versus time measured in the peroxidase substrate in the determination of the factor XIII activity described in Example 1
  • FIG. 2 shows a calibration curve for determining the factor XIII activity in plasma samples according to Example 2, in which the factor XIII activity is given as a percentage based on 100% pool plasma.
  • B-X-NHS is dissolved in 30% dimethylformamide.
  • the fibrinogen solution has a concentration of 2 mg / ml.
  • the molar ratio of biotin: fibrinogen used is 5: 1 during the conjugation. After 4 hours of reaction time, 60% of the B-X-NHS is coupled, i.e. one fibrin molecule carries 2 to 3 biotin molecules.
  • sample solution plasma, cell lysates, tissue extracts
  • biotin fibrinogen 100 ⁇ l of sample solution (plasma, cell lysates, tissue extracts) are placed in each well of the microtiter plate prepared according to B, 100 ⁇ l of biotin fibrinogen are added, mixed and the transamidation is started by adding 50 ⁇ l of beef trachino (5 NIHU / ml in 20 mM CaCl_) . After an incubation period of 5 minutes, all non-covalently bound biotin-fibrin molecules are washed out with a 4 M urea buffer.
  • a streptavidin-conjugated peroxidase is bound to the biotin-fibrin in a known manner and the change in the optical density of a solution of peroxidase substrate is measured.
  • the factor XIII activity of pool plasma samples is first determined in different dilution stages on the same microtiter plate on which the plasma samples to be examined are also measured - ⁇ -

Abstract

In order to determine the activity of transglutaminases, a solution of a physiological substrate of the transglutaminase to be determined marked with a marking substance is incubated with a solid-phase substrate of the enzyme in the presence of the specimen to be examined, the phases are separated and the amount of the marking susbtance present in the solid or in the liquid phase is taken as a measure of the transglutaminase activity.

Description

Verfahren zur Aktivitätsbestimmung von Transglut- aminasen Procedure for determining the activity of transglutaminases
B e s c h r e i b u n gDescription
Die Erfindung betrifft ein Verfahren zur Bestimmung der enzymatischen Aktivität von Transglutaminasen in Plasma, Zellen und Geweben durch Quervernetzung spezifischer markierter Proteinsubstrate und Verfahren zur Herstel¬ lung der markierten Substrate. Das erfindungsgemäße Bestimmungsverfahren kann angewendet werden, um bestimm¬ te Erkrankungen zu erkennen, einen Krankheitsverlauf zu überwachen oder eine Therapie zu kontrollieren.The invention relates to a method for determining the enzymatic activity of transglutaminases in plasma, cells and tissues by crosslinking specific labeled protein substrates and to methods for producing the labeled substrates. The determination method according to the invention can be used to detect certain diseases, to monitor a course of the disease or to control a therapy.
Als Transglutaminasen werden die Mitglieder einer Familie von Enzymen bezeichnet, von welchen bekannt ist, daß sie extrazellulär und intrazellulär Moleküle von verschiedenen Strukturproteinen durch enzymatische Knüpfung einer£-(^-Glutamyl)Lysyl-Bindung kovalent vernetzen (Übersichten: a. Lorand L. und Conrad, S.M., Transglutaminases, Molecular and Cellular Biochemistry 58: 9-35 (1984); b. Folk, J.E. und Finlayson, J.S., The £""(^-glutamyl)lysine crosslink and the catalytic role of transglutaminases, Advances in Protein Chemistry 31: 1-133 (1977).Transglutaminases are the members of a family of enzymes which are known to covalently crosslink extracellularly and intracellularly molecules of different structural proteins by enzymatic formation of a £ - (^ - glutamyl) lysyl bond (reviews: a. Lorand L. and Conrad, SM, Transglutaminases, Molecular and Cellular Biochemistry 58: 9-35 (1984); b. Folk, JE and Finlayson, JS, The £ "" (^ - glutamyl) lysine crosslink and the catalytic role of transglutaminases, Advances in Protein Chemistry 31: 1-133 (1977).
Das wichtigste Enzym der Transaminasenfamilie ist der Blutgerinnungsfaktor XIII. Als Substrate sind bekannt: Fibrin, Fibronectin, alpha-Plasmininhibitor, Thrombos- pondin, Collagen, alpha2-Makroglobulin, von Willebrand- Faktor, Faktor V, Aktin und Myosin.The most important enzyme in the transaminase family is blood coagulation factor XIII. The following are known as substrates: fibrin, fibronectin, alpha-plasmin inhibitor, thrombospondin, collagen, alpha 2 -macroglobulin, von Willebrand factor, factor V, actin and myosin.
Transglutaminasen sind beteiligt bei Blutgerinnung, Thrombosen, Regulation der Fibrinolyse, Verbrauchsko- agulopathie, Wundheilung, Tumormetastasierung, Arte- riosklerose, kongenitaler Faktor XIII-Mangel, Spontan¬ aborten, Rheuma, entzündlichen und immunologischen - Z -Transglutaminases are involved in blood coagulation, thrombosis, regulation of fibrinolysis, consumption coagulopathy, wound healing, tumor metastasis, arteriosclerosis, congenital factor XIII deficiency, spontaneous abortion, rheumatism, inflammatory and immunological - Z -
Erkrankungen. Daher besteht ein Bedarf an einer schnel¬ len und genauen Bestimmung dieser Enzyme.Diseases. There is therefore a need for a quick and precise determination of these enzymes.
Mehrere Verfahren zur Bestimmung von Faktor XIII sind bekannt:Several methods for determining factor XIII are known:
a) Bestimmung der Löslichkeit von Fibrin-Testgerinnseln durch verdünnte Säuren oder Harnstoff (Übersicht: Rasche, H. , Blutgerinnungsfaktor XIII und Fibrinsta¬ bilisierung. Klinische Wochenschrift 53_: 1137-1145 (1975)) . Diese Methoden jedoch messen nur semiquan¬ titativ und sind daher nur zur Diagnose bei ausge¬ prägtem Faktor XIII-Mangel anwendbar.a) Determination of the solubility of fibrin test clots by dilute acids or urea (overview: Rasche, H., blood coagulation factor XIII and fibrin stabilization. Clinical weekly 53_: 1137-1145 (1975)). However, these methods measure only semi-quantitatively and can therefore only be used for diagnosis in the case of a pronounced factor XIII deficiency.
b) Inkorporation von radioaktiven oder fluoreszierenden A inen in Casein. Faktor XIII activity using dansyl- cadavenin incorporation and gel filtration. Thrombo- sis Research 3 : 123-131 (1984); b) Musebeck, L., Polgar, J. , Fusus, L. , Kinetic determination of blood-coagulation Faktor XIII in plas a. Clinical Chemistry 31: 35-40 (1985)) .b) Incorporation of radioactive or fluorescent amines into casein. Factor XIII activity using dansyl-cadavenin incorporation and gel filtration. Thrombosis Research 3: 123-131 (1984); b) Musebeck, L., Polgar, J., Fusus, L., Kinetic determination of blood-coagulation factor XIII in plas a. Clinical Chemistry 31: 35-40 (1985)).
Diese Methoden messen präzise, sind aber sehr aufwendig, langwierig und nur für kleine Probenzahlen anwendbar.These methods measure precisely, but are very complex, lengthy and can only be used for small numbers of samples.
Der Erfindung liegt daher die Aufgabe zugrunde, ein Reagenz bzw. Verfahren zu entwickeln, um die enzymati- sche Aktivität von Transglutaminasen und insbesondere Faktor XIII spezifisch und quantitativ zu bestimmen. Der Test sollte einfach und schnell durchführbar sein und eine große Anzahl von Proben mit großer Genauigkeit parallel erfassen können.The object of the invention is therefore to develop a reagent or method to specifically and quantitatively determine the enzymatic activity of transglutaminases and in particular factor XIII. The test should be easy and quick to perform and should be able to record a large number of samples in parallel with great accuracy.
Gelöst wird diese Aufgabe erfindungsgemäß durch ein Verfahren zur Bestimmung der Aktivität von Transglutami- nasen, welches dadurch gekennzeichnet ist, daß man eine Lösung eines physiologischen Substrates der zu bestim¬ menden Transglutaminase, welches eine Markierung trägt, mit einem in fester Phase vorliegenden Substrat des Enzyms in Gegenwart der zu untersuchenden Probe inku¬ biert, die Phasen trennt und die in der festen oder in der flüssigen Phase vorliegende Markierung als Maß der Transglutaminase-Aktivität bestimmt.According to the invention, this object is achieved by a method for determining the activity of transglutami noses, which is characterized in that a solution of a physiological substrate of the transglutaminase to be determined, which carries a label, is incubated with a solid phase of the enzyme in the presence of the sample to be examined, the phases are separated and the Marking present in the solid or in the liquid phase is determined as a measure of the transglutaminase activity.
Als Transgluta inasesubstrat kann im Rahmen der Erfin¬ dung jedes physiologische Substrat der jeweils interes¬ sierenden Transglutaminase verwendet werden. Für die Bestimmung der physiologisch besonders wichtigen Trans¬ aminase Blutgerinnungsfaktor XIII eignen sich die oben erwähnten Substrate Fibrin, Fibronectin, alpha^Plasmin- inhibitor, Thrombospondin, Collagen, alpha2-Makroglobu- lin, von Willebrand-Faktor, Faktor V, Aktin und Myosin. Besonders bevorzugt wird Fibrin als in situ-Substrat in Form von Fibrinogen, aus dem während der Bestimmung durch Thrombineinwirkung Fibrin entsteht, verwendet.In the context of the invention, any physiological substrate of the transglutaminase of interest in each case can be used as the transglutinase substrate. The substrates fibrin, fibronectin, alpha ^ plasmin inhibitor, thrombospondin, collagen, alpha 2 macroglobulin, von Willebrand factor, factor V, actin and myosin are suitable for determining the physiologically particularly important trans-aminase blood coagulation factor XIII . Fibrin is particularly preferably used as an in situ substrate in the form of fibrinogen, from which fibrin is formed during the determination by the action of thrombin.
In der flüssigen wie in der festen Phase wird vorzugs¬ weise das gleiche Enzymsubstrat verwendet, obwohl dies nicht erfindungswesentlich ist und es allein darauf ankommt, daß das Substrat von dem jeweils zu bestimmen¬ den Enzym überhaupt als Substrat verwendet wird. Im Rahmen des erfindungsgemäßen Verfahrens verknüpft das Enzym in einer Art Vernetzungsreaktion die beiden Substrate, das in der flüssigen Phase vorliegende markierte Substrat und das an der festen Phase gebun¬ dene Substrat, miteinander. Nach Trennung der Phasen kann das durch die Vernetzungsreaktion an die feste Phase gebundene markierte Substrat anhand seiner Mar¬ kierung bestimmt werden. Die Menge an vernetzten! mar¬ kiertem Substrat korreliert dabei der gesuchten Enzym- - h -The same enzyme substrate is preferably used in the liquid as in the solid phase, although this is not essential to the invention and the only thing that matters is that the substrate of the enzyme to be determined is used as substrate at all. In the context of the method according to the invention, the enzyme combines the two substrates, the labeled substrate present in the liquid phase and the substrate bound to the solid phase, in a type of crosslinking reaction. After separation of the phases, the labeled substrate bound to the solid phase by the crosslinking reaction can be determined on the basis of its marking. The amount of connected! marked substrate correlates the sought enzyme - H -
aktivität. Setzt man dabei eine bekannte Menge an markiertem gelöstem Substrat ein, so kann auch die in Lösung verbliebene Substratmenge als Maß für die Be¬ stimmung der Enzymaktivität herangezogen werden. Zur Markierung geeignet sind beispielsweise radioaktive Isotope wie z.B. Jodisotope, Farbstoffe, Fluoreszenz- farbstoffe, Enzyme, sowie die Partner von physiologi¬ schen Bindungspaaren wie z.B. Biotin/Streptavidin oder Antigen/Antikörper. Bevorzugt wird im Rahmen der Erfin¬ dung als Markierung einer der Partner des Bindepaares Biotin/Streptavidin. Der Nachweis kann dann in an sich bekannter Weise mit dem anderen Bindepartner, der in eine bestimmbare Form gebracht wird, erfolgen, bei¬ spielsweise durch Kupplung an ein bestimmbares Enzym wie Peroxidase. Daher wird bevorzugt im Rahmen der Faktor XII-Bestimmung als markiertes lösliches Substrat Biotin/Fibrinogen verwendet und mit einem Peroxidase- Streptavidin-Konjugat unter Anwendung der üblichen Peroxidase-Bestimmungsmethode nachgewiesen. Die Bestim¬ mung von Peroxidase über Farbreaktionen ist dem Fach¬ mann geläufig und bedarf hier keiner näheren Beschrei¬ bung.activity. If a known amount of labeled dissolved substrate is used, the amount of substrate remaining in solution can also be used as a measure for determining the enzyme activity. Radioactive isotopes such as e.g. Iodine isotopes, dyes, fluorescent dyes, enzymes, as well as the partners of physiological binding pairs such as e.g. Biotin / streptavidin or antigen / antibody. In the context of the invention, preference is given to marking one of the partners of the binding pair biotin / streptavidin. The detection can then be carried out in a manner known per se with the other binding partner, which is brought into a determinable form, for example by coupling to a determinable enzyme such as peroxidase. Therefore, biotin / fibrinogen is preferably used as the labeled soluble substrate in the context of factor XII determination and detected with a peroxidase-streptavidin conjugate using the usual peroxidase determination method. The determination of peroxidase via color reactions is familiar to the person skilled in the art and does not require any further description here.
Zur Markierung der gelösten Substrate mittels Biotin hat sich besonders Biotin-<-Aminocapronsäure-N-hydroxy- succinimid-Ester (B-X-NHS) als vorteilhaft erwiesen, da es die biologische Aktivität der empfindlichen Substrate wie z.B. Fibrinogen aufrecht erhält. Für die Verwendung als Substrat wird Fibrinogen zweckmäßig in gereinigter Form verwendet. Zur Reinigung eignet sich insbesondere das Verfahren gemäß Mahn, I. und Müller-Berghaus, G., Studies on catabolism of 125-1-labelled fibrinogen in normal rabbits and in rabbits with indwelling intra- venous catheters: Methodologie aspects. Haemostasis 4_: 40-50 (1975)). - F -Biotin - <- aminocaproic acid-N-hydroxysuccinimide ester (BX-NHS) has proven to be particularly advantageous for labeling the dissolved substrates by means of biotin, since it maintains the biological activity of the sensitive substrates such as fibrinogen. Fibrinogen is expediently used in a purified form for use as a substrate. The method according to Mahn, I. and Müller-Berghaus, G., Studies on catabolism of 125-1-labeled fibrinogen in normal rabbits and in rabbits with indwelling intravenous catheters: Methodology aspects is particularly suitable for cleaning. Haemostasis 4_: 40-50 (1975)). - F -
Wird beim Verfahren der Erfindung die an die feste Phase durch die Vernetzungsreaktion gebundene Markie¬ rung bestimmt, so .hat es sich als vorteilhaft erwiesen, die feste Phase mit einer gepufferten Lösung einer chaotropen Substanz, insbesondere mit einer Harnstoff¬ lösung oder mit verdünnter Säure von nicht gebundenem löslichen Substrat zu reinigen.If, in the process of the invention, the marking bound to the solid phase by the crosslinking reaction is determined, it has proven to be advantageous to coat the solid phase with a buffered solution of a chaotropic substance, in particular with a urea solution or with dilute acid clean unbound soluble substrate.
Als feste Phase und Träger des in fester Phase vorlie¬ genden Substrates eignen sich die üblicherweise für Enzymreaktionen geeigneten Substrate wie z.B. Träger¬ material auf Basis von Kohlenhydraten wie Agarose, Cellulose, Stärke oder dgl. oder auf Basis von Kunst¬ stoffen, wie sie insbesondere für Mikrotiterplatten verwendet werden. Die Bindung des Substrates an den festen Träger kann adsorptiv, durch Ausbildung einer kovalenten Bindung mit dem Substrat oder durch Vernet¬ zung des Substrats auf der Trägeroberfläche erfolgen. Aufgrund der Einfachheit der Herstellung und der unmit¬ telbaren Anwendbarkeit für das erfindungsgemäße Verfah¬ ren eignet sich besonders die adsorptive Bindung an Mikrotiterplatten.Suitable substrates for the solid phase and carrier of the substrate present in the solid phase are those which are usually suitable for enzyme reactions, such as e.g. Carrier material based on carbohydrates such as agarose, cellulose, starch or the like or based on plastics as used in particular for microtiter plates. The substrate can be bound to the solid support by adsorption, by forming a covalent bond with the substrate or by crosslinking the substrate on the support surface. Because of the simplicity of production and the direct applicability for the method according to the invention, adsorptive binding to microtiter plates is particularly suitable.
Um unspezifische Bindungen von löslichem Substrat zu unterdrücken, wird die Trägeroberfläche vorteilhaft mit einem nicht quervernetzbaren unspezifischen Protein wie Rinderserumalbumin oder Milchprotein behandelt bzw. beschichtet.In order to suppress non-specific binding of soluble substrate, the carrier surface is advantageously treated or coated with a non-crosslinkable non-specific protein such as bovine serum albumin or milk protein.
Ein weiterer Gegenstand der Erfindung ist ein Reagenz zur Bestimmung der Aktivität von Transglutaminasen, welches dadurch gekennzeichnet ist, daß es ein festpha- sengebundenes physiologisches Transglutaminasesubstrat und ein wasserlösliches markiertes physiologisches Transglutaminasesubstrat enthält. - & -Another object of the invention is a reagent for determining the activity of transglutaminases, which is characterized in that it contains a solid-phase-bound physiological transglutaminase substrate and a water-soluble labeled physiological transglutaminase substrate. - & -
Das festphasengebundene Substrat wird zweckmäßigerweise hergestellt unter Verwendung einer Lösung, die etwa 0,1 bis 10 mg/1 Substrat enthält und mit dem festen Träger kontaktiert wird unter Bindung des Substrats. Bei adsorptiver Bindung am Träger führt dies zu einer ausreichenden Beladung desselben. Bei Mikrotiterplatten als Träger und adsorptiver Bindung ergaben 0,5 bis 5 μg Substrat/ Napf (well) gute Ergebnisse. Das lösliche markierte Substrat wird allgemein in einer Konzentration von 0,05 bis 10 mg/ml, vorzugsweise 0,5 bis 2 mg/ml eingesetzt.The solid-phase-bound substrate is expediently prepared using a solution which contains about 0.1 to 10 mg / l of substrate and is contacted with the solid support with binding of the substrate. With adsorptive binding to the carrier, this leads to an adequate loading of the carrier. With microtiter plates as supports and adsorptive binding, 0.5 to 5 μg substrate / well (well) gave good results. The soluble labeled substrate is generally used in a concentration of 0.05 to 10 mg / ml, preferably 0.5 to 2 mg / ml.
Ein bevorzugtes Reagenz, welches für die Aktivitätsbe¬ stimmung von Faktor XIII geeignet ist, enthält an eine feste Phase gebundenes Fibrinogen, markiertes lösliches Fibrinogen und Thrombin.A preferred reagent, which is suitable for determining the activity of factor XIII, contains fibrinogen bound to a solid phase, labeled soluble fibrinogen and thrombin.
In einer besonders bevorzugten Ausführungsform enthält dieses Reagenz als lösliches Substrat Biotin-Fibrinogen, sowie, vor Gebrauch davon getrennt, Streptavidin-Peroxi- dase-Konjugat und ein NachweisSystem für Peroxidase.In a particularly preferred embodiment, this reagent contains, as soluble substrate, biotin fibrinogen, and, before use, separately streptavidin-peroxidase conjugate and a detection system for peroxidase.
Durch die Erfindung wird eine einfache, rasch durchführ¬ bare Bestimmung der Transglutaminasen-Aktivität ermög¬ licht, die für die Diagnose und die Therapie einer mit den Transglutaminasen verbundenen Erkrankungen vorteil¬ haft angewendet werden kann.The invention enables a simple, rapid determination of the transglutaminase activity, which can be used advantageously for the diagnosis and therapy of diseases associated with the transglutaminases.
Die folgenden Beispiele erläutern die Erfindung in Verbindung mit der beigefügten Zeichnung weiter. In dieser stellen dar:The following examples further illustrate the invention in conjunction with the accompanying drawing. In this represent:
Figur 1 eine graphische Darstellung der im Peroxidase- substrat gemessenen Adsorption gegen die Zeit bei der in Beispiel 1 beschriebenen Bestimmung der Faktor XIII-Aktivität, Figur 2 eine Eichkurve zur Bestimmung der Faktor XIII- Aktivität in Plasmaproben gemäß Beispiel 2, bei der die Faktor XIII-Aktivität als Prozent bezogen auf 100 % Poolplasma angegeben ist.FIG. 1 shows a graphic representation of the adsorption versus time measured in the peroxidase substrate in the determination of the factor XIII activity described in Example 1, FIG. 2 shows a calibration curve for determining the factor XIII activity in plasma samples according to Example 2, in which the factor XIII activity is given as a percentage based on 100% pool plasma.
B e i s p i e l 1Example 1
Bestimmung der Faktor XIII-AktivitätDetermination of factor XIII activity
A) Herstellung eines Biotin-Fibrinogen-Konjugates als lösliches SubstratA) Preparation of a biotin-fibrinogen conjugate as a soluble substrate
B-X-NHS wird in 30 % Dimethylformamid gelöst. Die Fibrinogenlösung hat eine Konzentration von 2 mg/ml.B-X-NHS is dissolved in 30% dimethylformamide. The fibrinogen solution has a concentration of 2 mg / ml.
Gleiche Volumenteile der beiden Lösungen werden zusam¬ mengegeben und reagieren 4 Stunden bei 21°C. In dieser Zeit erfolgt die Verknüpfung von Biotin-X-Molekülen mit den Aminogruppen des Fibrinogens. Die Lösung biotini- lierten Fibrinogens wird anschließend bei 4°C über Nacht gegen einen phosphathaltigen Puffer dialysiert. Nicht konjugiertes B-X-NHS wird durch Gelfiltration auf Sephadex G 25 entfernt.The same parts by volume of the two solutions are added together and react for 4 hours at 21 ° C. During this time, biotin-X molecules are linked to the amino groups of the fibrinogen. The solution of biotinylated fibrinogen is then dialyzed against a phosphate-containing buffer at 4 ° C. overnight. Unconjugated B-X-NHS is removed by gel filtration on Sephadex G 25.
Das eingesetzte molare Verhältnis Biotin:Fibrinogen beträgt 5:1 während der Konjugation. Nach 4 Stunden Reaktionszeit sind 60 % des B-X-NHS gekoppelt, d.h. ein Fibrinmolekül trägt 2 bis 3 Biotinmoleküle.The molar ratio of biotin: fibrinogen used is 5: 1 during the conjugation. After 4 hours of reaction time, 60% of the B-X-NHS is coupled, i.e. one fibrin molecule carries 2 to 3 biotin molecules.
Niedere oder höhere Konjugationszeiten sowie Veränderun¬ gen des molaren Verhältnisses Biotin:Fibrinogen haben sich als weniger günstig erwiesen. - g -Lower or higher conjugation times and changes in the molar ratio of biotin: fibrinogen have proven to be less favorable. - g -
B) Herstellung des in fester Phase vorliegenden Sub¬ stratsB) Production of the substrate present in the solid phase
Pro Testansatz werden 20 μl einer Fibrinogenlösung mit 1 mg/ml für 2 Std. auf Mikrotiterplatten adsorbiert. Nach einem Waschschritt mit Kalzium-haltigem Puffer werden freie Bindungsstellen mit 5 % Rinder-Serum-Albu¬ min für 2 Stunden abgesättigt und dann nochmals gewa¬ schen.For each test batch, 20 μl of a fibrinogen solution with 1 mg / ml are adsorbed on microtiter plates for 2 hours. After a washing step with calcium-containing buffer, free binding sites are saturated with 5% bovine serum albumin for 2 hours and then washed again.
C) Durchführung der AktivitätsbestimmungC) Carrying out the activity determination
100 μl Probelösung (Plasma, Zellysate, Gewebeextrakte) werden in jede Vertiefung der nach B hergestellten Mikrotiterplatte vorgelegt, 100 μl Biotin-Fibrinogen zugegeben, gemischt und die Transamidierung durch Zugabe von 50 μl Rinderthro bin (5 NIHU/ml in 20 mM CaCl_) gestartet. Nach 5 minütiger Inkubationszeit werden alle nicht kovalent gebundenen Biotin-Fibrin- Moleküle durch einen 4 M Harnstoffpuffer ausgewaschen. Zu Detektierung der verbliebenen Biotin-Fibrin-Moleküle wird eine Streptavidin-konjugierte Peroxidase in bekann¬ ter Weise am Biotin-Fibrin gebunden und die Änderung der optischen Dichte einer Lösung von Peroxidasesubstrat gemessen.100 μl of sample solution (plasma, cell lysates, tissue extracts) are placed in each well of the microtiter plate prepared according to B, 100 μl of biotin fibrinogen are added, mixed and the transamidation is started by adding 50 μl of beef trachino (5 NIHU / ml in 20 mM CaCl_) . After an incubation period of 5 minutes, all non-covalently bound biotin-fibrin molecules are washed out with a 4 M urea buffer. To detect the remaining biotin-fibrin molecules, a streptavidin-conjugated peroxidase is bound to the biotin-fibrin in a known manner and the change in the optical density of a solution of peroxidase substrate is measured.
B e i s p i e l 2Example: 2
Faktor XIII-Bestimmung in humanen PlasmaprobenFactor XIII determination in human plasma samples
Auf der gleichen Mikrotiterplatte, auf der auch die zu untersuchenden Plasmaproben gemessen werden, erfolgt zunächst die Bestimmung der Faktor XIII-Aktivität von Poolplasmaproben in unterschiedlichen Verdünnungsstufen - β -The factor XIII activity of pool plasma samples is first determined in different dilution stages on the same microtiter plate on which the plasma samples to be examined are also measured - β -
(molares Verhältnis Poolplasma:Verdünnungspuffer 1:10, 1:15, 1:20 und 1:30). Trägt man die Extinktionswerte der Proben gegen die Meßzeit in einem Diagramm auf, ergeben sich Kurvenverläufe, deren Absolutwert umso höher ist, je konzentrierter das jeweils verwendete Poolplasma war (Fig. 1) . Zur Gewinnung einer Eichkurve werden in einem weiteren Diagramm die Extinktionswerte zum Zeitpunkt 120 Minuten gegen das Verdünnungsverhält¬ nis der Proben aufgetragen (Fig. 2) . Durch Ablesen der 120 Minuten-Werte der im molaren Verhältnis Plasmapro¬ be:Verdünnungspuffer 1:10 verdünnten Plasmaproben erhält man Prozentwerte der in der jeweiligen Probe enthaltenen Thrombin-induzierbaren Faktor XIII-Aktivi¬ tät bezogen auf das verwendete Poolplasma. (molar ratio pool plasma: dilution buffer 1:10, 1:15, 1:20 and 1:30). If the extinction values of the samples are plotted against the measuring time in a diagram, curve profiles are obtained, the absolute value of which is higher the more concentrated the pool plasma used was (FIG. 1). To obtain a calibration curve, the extinction values at 120 minutes are plotted against the dilution ratio of the samples (FIG. 2). By reading the 120 minute values of the plasma samples diluted in a molar ratio of plasma sample: dilution buffer 1:10, percentages of the thrombin-inducible factor XIII activity contained in the respective sample are obtained based on the pool plasma used.

Claims

-40 -P a t e n t a n s p r ü c h e -40 -P claims
1. Verfahren zur Bestimmung der Aktivität von Trans¬ glutaminasen, d a d u r c h g e k e n n z e i c h n e t , daß man eine Lösung eines physiologischen Substra¬ tes der zu bestimmenden Transglutaminase, welches eine Markierung trägt, mit einem in fester Phase vorliegenden Substrat des Enzyms in Gegenwart der zu untersuchenden Probe inkubiert, die Phasen trennt und die in der festen oder in der flüssigen Phase vorliegende Markierung als Maß der Trans- glutaminase-Aktivität bestimmt.1. A method for determining the activity of transglutaminases, characterized in that a solution of a physiological substrate of the transglutaminase to be determined, which carries a label, is incubated with a solid phase of the enzyme in the presence of the sample to be examined, the phases are separated and the marking present in the solid or in the liquid phase is determined as a measure of the transglutaminase activity.
2. Verfahren nach Anspruch 1, d a d u r c h g e k e n n z e i c h n e t , daß man in flüssiger und in fester Phase das gleiche Enzymsubstrat verwendet.2. The method of claim 1, d a d u r c h g e k e n n z e i c h n e t that the same enzyme substrate is used in the liquid and in the solid phase.
3. Verfahren nach Anspruch 1 oder 2, d a d u r c h g e k e n n z e i c h n e t , daß man als Substrat für die Transglutaminase Faktor XIII Fibrinogen verwendet und in Gegenwart von Thrombin inkubiert.3. The method of claim 1 or 2, d a d u r c h g e k e n n z e i c h n e t that one uses as a substrate for the transglutaminase factor XIII fibrinogen and incubated in the presence of thrombin.
4. Verfahren nach einem der vorhergehenden Ansprüche, d a d u r c h g e k e n n z e i c h n e t , daß man als Markierung einen Partner des Bindepaa¬ res Biotin/Streptavidin verwendet und die Markie¬ rung mit einem Konjugat aus dem anderen Bindungs¬ partner und einem nachweisbaren Enzym bestimmt. 4. The method according to any one of the preceding claims, characterized in that one uses a partner of the Bindepaa¬ res biotin / streptavidin and the Markie¬ tion with a conjugate of the other binding partner and a detectable enzyme determined.
5. Verfahren nach Anspruch 4, d a d u r c h g e k e n n z e i c h n e t , daß man als Markierungsenzym Peroxidase verwendet.5. The method of claim 4, d a d u r c h g e k e n n z e i c h n e t that peroxidase is used as the labeling enzyme.
6. Verfahren nach einem der vorhergehenden Ansprüche, d a d u r c h g e k e n n z e i c h n e t , daß man die an die feste Phase gebundene Markierung bestimmt und nicht kovalent gebundenes markiertes Substrat durch eine gepufferte Lösung chaotroper Substanzen, insbesondere eine Harnstofflösung oder verdünnte Säure von der festen Phase entfernt.6. The method according to any one of the preceding claims, d a d u r c h g e k e n n z e i c h n e t that the label bound to the solid phase is determined and the non-covalently bound labeled substrate is removed from the solid phase by a buffered solution of chaotropic substances, in particular a urea solution or dilute acid.
7. Reagenz zur Bestimmung der Aktivität von Transglut¬ aminasen, d a d u r c h g e k e n n z e i c h n e t , daß es ein festphasengebundenes physiologisches Transglutaminasesubstrat und ein wasserlösliches markiertes physiologisches Transglutaminasesub¬ strat enthält.7. Reagent for determining the activity of transglutaminases, that is, that it contains a solid-phase-bound physiological transglutaminase substrate and a water-soluble labeled physiological transglutaminase substrate.
8. Reagenz nach Anspruch 7, d a d u r c h g e k e n n z e i c h n e t , daß es zum Nachweis von Faktor XIII an eine feste Phase gebundenes Fibrinogen, markiertes lösliches Fibrinogen und Thrombin enthält.8. A reagent according to claim 7, which also contains fibrinogen bound to a solid phase, labeled soluble fibrinogen and thrombin for the detection of factor XIII.
9. Reagenz nach Anspruch 8, d a d u r c h g e k e n n z e i c h n e t , daß das lösliche markierte Fibrinogen ein Fibrino- gen-Biotin-Konjugat ist und getrennt davon Strept- avidin-konjugierte Peroxidase und Peroxidasesub¬ strat enthält. 9. Reagent according to claim 8, that the soluble labeled fibrinogen is a fibrinogen-biotin conjugate and contains streptavidin-conjugated peroxidase and peroxidase substrate separately therefrom.
PCT/EP1989/001169 1988-10-07 1989-10-06 Process for determining the activity of transglutaminases WO1990004038A1 (en)

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