WO1992010761A1 - Procede et reactifs pour la determination de l'activite enzymatique des transglutaminases (ec 2.3.2.13) - Google Patents

Procede et reactifs pour la determination de l'activite enzymatique des transglutaminases (ec 2.3.2.13) Download PDF

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WO1992010761A1
WO1992010761A1 PCT/EP1991/002223 EP9102223W WO9210761A1 WO 1992010761 A1 WO1992010761 A1 WO 1992010761A1 EP 9102223 W EP9102223 W EP 9102223W WO 9210761 A1 WO9210761 A1 WO 9210761A1
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transglutaminase
solution
antibody
group
thrombin
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German (de)
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Carl-Erik Dempfle
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    • 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
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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 present invention relates to a new method for determining the activity of the enzymes of the enzyme commission number EC 2.3.2.13, in particular the activity of the blood coagulation factor XIII (plasma transglutaminase), and a reagent kit for carrying out the determination.
  • the enzymes of the enzyme commission number EC 2.3.2.13 in particular the activity of the blood coagulation factor XIII (plasma transglutaminase), and a reagent kit for carrying out the determination.
  • Blood coagulation is a complex, multi-phase process that can be triggered by various physiological and pathological processes and is mainly used to stop bleeding in vivo.
  • Various so-called coagulation factors are involved in blood coagulation.
  • Most of these are proteases that activate them by means of highly specific proteolytic effects on other coagulation factors, or cofactors of these reactions.
  • the aim of these reactions is to convert the fibrinogen into polymeric fibrin.
  • Both fibrinogen and factor XIII experience a proteolytic cleavage of polypeptides through the enzyme thrombin, which arises from prothrombin in the course of blood coagulation. After these polypeptides have been split off, fibrinogen is formed from fibrin monomer, which polymerizes spontaneously.
  • Cleavage of the polypeptide from the catalytic unit of factor XIII causes the activation of the enzyme.
  • the catalytically inactive proenzyme factor XIII is found in blood plasma and in platelets. Active factor XIII catalyzes the formation of covalent bonds between the polymerized fibrin molecules and thus an increase in the mechanical resistance of the fibrin.
  • pathophysiologically important molecules such as fibronectin and alpha-2-antiplasmin are covalently coupled to the fibrin clot. The coupling of fibronectin enables cells to grow into the fibrin clot, the coupling of alpha-2-antiplasmin leads to an increase in the resistance of the fibrin to proteases such as plasmin.
  • Transglutaminases are also found in numerous tissues and cells, such as erythrocytes, leucocytes, endothelial cells, liver cells, various tumor cells, etc. These intracellular transglutaminases show similar specificities to factor XHI, but their activity is not dependent on thrombin. The known detection methods for measuring the transglutaminase activity have been developed on the basis of the protein transglutaminase activity of factor XIII.
  • the activity of factor XIII and other transglutaminases can be determined by gel electrophoresis on agarose gel plates and subsequent reaction with strongly colored or fluorescent cadaverine derivatives, casein and thrombin.
  • the excess cadaverine has to be removed from the gel plate by relatively cumbersome fixation and washing. The measurement is carried out by densitometric evaluation of the gel plates (Lorand et al. (Anal. Biochem. 131, pages 419-425 (1983)).
  • the adsorbed galactosidase activity is determined by the release of p-nitrophenol from p-nitrophenyl- ⁇ -D-galactopyranoside and forms a measure of the concentration of factor XIII. This method is also still very complex and inaccurate due to the different transmission steps.
  • the object was therefore to find a simple analytical method for determining the blood coagulation factor XIII (plasma transglutaminase and thrombocyte transglutaminase) and other transglutaminases of the enzyme group EC 2.3.2.13, using as few reagents and reaction and washing steps as possible can be automated as far as possible and can be carried out by untrained specialists.
  • blood coagulation factor XIII plasma transglutaminase and thrombocyte transglutaminase
  • other transglutaminases of the enzyme group EC 2.3.2.13 using as few reagents and reaction and washing steps as possible can be automated as far as possible and can be carried out by untrained specialists.
  • microtiter plates are initially coated with the protein or polypeptide, preferably with casein, this can be used in optimum concentration, so that all active sites on the microtiter plate to which such proteins or polypeptides can attach are blocked. This saves the step necessary in the method of 'Velasco' and 'Lee' to initially block free active centers of the microplate with milk proteins between the accumulation of the casein and the reaction with the antibodies.
  • the surface is such that proteins and peptides are sufficiently non-covalently bound.
  • those made of other plastics such as polyethylene, polycarbonate or polypropylene, can also be used as reaction vessels. If these do not sufficiently adsorb the required proteins and peptides, the surface can additionally be activated by glutaraldehyde, cyanogen bromide or other activators.
  • the shape of the reaction and measuring vessels is of course completely variable, for example plastic test tubes or centrifuge tubes or photometer cuvettes of various shapes can be used.
  • the simplest and cheapest is to carry out the tests with commercially available microtiter plates, for example with 96 wells, each with a volume of 300-400 ⁇ l, or microtiter strips with 8, 12 or 16 wells, also with a volume of 300-400 ⁇ l, since these are suitable for the test of series trials are particularly suitable.
  • Casein in particular is used as the protein, since both factor XIII and tissue and cell transglutaminases react with this substrate.
  • substrates containing glutamine can also be used in the same way.
  • natural factor XIII or transglutaminase substrates such as fibrinogen, collagen, fibronectin, thrombospondin, etc., as well as derivatives and fragments of these proteins as well as natural and synthetic peptides which contain the glutamine necessary for the reaction can be used.
  • the prerequisite is that the substances are absolutely free of transglutamines, which is not always guaranteed with native fibrinogen and fibronectin. Since the substrates containing glutamine differ in their reactivity with the transglutaminases and in their ability to bind to the plastic surfaces of the reaction vessels, it is necessary to carry out control samples with known activity when measuring the transglutaminase activity.
  • the glutamine-containing substrate can be bound to the reaction vessel (di microtiter plate) either shortly before the determinations are actually carried out, but the coated plates can also be manufactured and stored under sterile conditions.
  • Particularly suitable for the reactive primary amine of the formula I are those which have a linear methylene chain with 4 to 8 carbon atoms, with 5 to 6 carbon atoms being particularly preferred. With shorter chains, a reaction is obviously no longer possible for steric reasons; with longer chains, the reaction rate drops sharply. Practically all groups which do not interfere with the transglutaminase reaction, but which enter into an antibody reaction, are suitable as substituents -X- of formula (I). Larger, optionally substituted carbocyclic groups are particularly suitable, although a certain hydrophobicity seems to increase the reactivity of the nucleophiolic primary amino group.
  • the substrates known from the literature dinitrophenylcadaver, biotinamido-pentylamine and dansylcadaverine, are therefore suitable, for example.
  • polyclonal antibodies can also be used, provided they are sufficiently specific for the above substrates and are available in a sufficiently pure form so that coupling with an indicator enzyme is possible.
  • polyclonal antisera can be purified by affinity chromatography or ion exchange chromatography.
  • a control reaction with factor XIII-free plasma or trans glutaminase-free sample can be checked that such polyclonal antibodies have no reactivity with the normal plasma or sample components or components of the coating solutions de reaction vessels.
  • An enzyme is preferably used as a label for the antibodies used, in order to increase the sensitivity accordingly.
  • the markings commonly used in technology with alkaline phosphatase, peroxidase or urease can also be used advantageously for this test.
  • Corresponding detection reagents are known, for example p-nitrophenylphosphate for alkaline phosphatase, 2,2'-azino-bis- (3-ethylbenzthiazolin-6-sulphonic acid) or o-phenylenediamine for peroxidase and bromcresol purple for urease.
  • markings with chromophoric, in particular fluorescent groups can also be used, as a result of which the additional detection reactions remain and the reaction can be monitored directly photometrically.
  • Radioactive markings are in many laboratories because of the associated safety precautions and the outlay on equipment not “desired, but of course the method according to the invention can also be carried out by radioactive labeling of either the primary amine used or the antibody used. In the former case, the step of antibody labeling of the reaction product can already be omitted, in the second case only afterwards a radioactivity measurement of the bound substrates is carried out at this marker and the washout via excess antibody.
  • the method according to the invention has been worked out in particular for the detection of the blood coagulation factor XIII (plasma transglutaminase), but the other enzymes of the number EC 2.3.2.13 can also be determined in this way.
  • microtiter plates with 96 wells of 400 ⁇ l of polystyrene are incubated at 37 ° C. with a solution of casein in 0.1 M phosphate buffer pH 8.0 or 0.05 M tris (hydroxymethyl) aminomethane and 0.1 M NaCl, pH 8.5.
  • the optimal concentration of casein is between 3 and 6 ⁇ g / ml solution, the incubation time at 37 ° C. should be at least 2 hours and at most 24 hours.
  • the microtiter plates are then emptied and rinsed with a buffer solution, for example with a buffered NaCl solution.
  • Figures 1 and 2 The effect of casein concentration in the casein solution on the sensitivity of the test for two types of polystyrene microtiter plates with and without glutaraldehyde activation is shown in Figures 1 and 2.
  • Optimal sensitivity is achieved in a concentration range of 1-10, preferably 3-6, ⁇ g casein per ml buffer solution.
  • Figure 3 shows comparative experiments with casein and the proteolytic fibrinogen fragment D as coating material. Both Ca and fibrinogen fragment D show a sensitive reaction proportional to the transglutaminase activity of factor XIII.
  • the measurements for FIGS. 1, 2 and 3 were carried out according to Examples 2 and 3 with standardized samples of known factor XHI activity.
  • a buffer solution is made from:
  • the samples used are diluted with the sample dilution buffer, a dilution between 1:15 and 1:40, in particular 1:20, being used to measure the factor XIII activity in human blood plasma. Different dilution factors result for other samples.
  • a buffer stock solution is made from:
  • the active amine substrate solution is made from:
  • DNPC dinitrophenylcadaverine
  • a microtiter plate (96 wells) according to Example 1 is rinsed with solution b).
  • 100 ⁇ l of diluted sample, 100 ⁇ l of substrate solution c) and 100 ⁇ l of thrombin solution are poured into each well (2 to 3 parallel tests per sample and control with standard plasma with a known transglutaminase concentration and factor X ⁇ i-deficient plasma).
  • the reagent supernatant is rinsed out with solution b).
  • Microtiter plates according to the above example are then connected to the substrate reaction with 250 ⁇ l / well of an anti-dinitrophenyl-antibody conjugate solution from 25 ⁇ l antibody conjugate (phosphatase marked) and 25 ml of solution b) and incubated for 60 minutes at room temperature.
  • the antibody-enzyme conjugate is produced by glutaraldehyde coupling from commercially available antibody (Sigma, Deisenhofen) and commercially available enzyme alkaline phosphatase (Boehringer, Mannheim) according to a method described by Harlow and Lane (Harlow E, Lane D, Antibodies: A laboratory manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA (1988) Other methods for coupling antibodies and enzymes can also be used in the described method.
  • microtiter plate is washed with solution b) and mixed with 250 ⁇ l phosphatase substrate solution per well.
  • phosphate substrate solution To prepare the phosphate substrate solution, mix: 5 ml of 1 M diethanolamine, 0.01 M magnesium chloride, pH 9.5 15 mg of p-nitrophenyl phosphate (substrate tablet, e.g. from Sigma, Deisen ⁇ ofen), 20 ml of distilled water.
  • substrate tablet e.g. from Sigma, Deisen ⁇ ofen
  • the absorption is then measured photometrically at 405 nm.
  • the measurement can be carried out kinetically by measurements after defined incubation times, preferably after 10, 20, 30, 40, 50 and 60 minutes, or by end point measurement after adding a stop reagent, preferably 50 ⁇ l per well of a 3 M NaOH solution.
  • the factor XIII activity is calculated by comparison with the absorption of the controls. A corresponding calibration curve is shown in Figure 4.

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Abstract

L'invention concerne un procédé pour la détermination du facteur de coagulation XIII (plasma-transglutaminase et thrombocytes-transglutaminase) et d'autres transglutaminases du groupe enzymatique EC 2.3.2.13, procédé selon lequel: a) des plaques de microtitrage en matière plastique sont garnies, en surface, de protéines ou de peptides renfermant de la glutamine; b) du chlorure de calcium, un composé thio agissant comme réducteur, un composé présentant un groupe amino primaire de formule NH2-(CH2)n-X dans laquelle n est un nombre entier compris entre 4 et 8, et X désigne un groupe inerte susceptible de s'associer à des anticorps spécifiques, ainsi qu'une prise d'essai contenant de la transglutaminase, sont amenés à réagir sur la plaque garnie de microtitrage, réaction au cours de laquelle de la thrombine est éventuellement ajoutée en vue de l'activation protéolytique de de la transglutaminase; c) l'amine liée à la plaque, sous l'action de la transglutaminase, est mise à réagir sur le groupe X avec un anticorps marqué; d) la quantité d'amine liée, ou éventuellement d'anticorps, est déterminée à l'aide d'un réactif indicateur approprié; e) les restes de réactifs dissous étant éliminés par lavage entre chacune des différentes étapes. L'invention concerne également un jeu de réactifs pour la mise en oeuvre de ce procédé.
PCT/EP1991/002223 1990-12-06 1991-11-26 Procede et reactifs pour la determination de l'activite enzymatique des transglutaminases (ec 2.3.2.13) Ceased WO1992010761A1 (fr)

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DE19904038899 DE4038899C2 (de) 1990-12-06 1990-12-06 Verfahren und Reagenzien zur Bestimmung der enzymatischen Aktivität von Transglutaminasen (EC 2.3.2.13)
DEP4038899.9 1990-12-06

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998058078A1 (fr) * 1997-06-18 1998-12-23 Korea Green Cross Corporation Procede permettant de determiner l'activite enzymatique du facteur xiii de coagulation sanguine a l'aide d'un substrat consistant en un monomere de fibrine purifie
WO2001029090A1 (fr) * 1999-10-20 2001-04-26 Immco Diagnostics Methode de dosage immunologique servant a detecter des anticorps d'une maladie coeliaque
US6703208B1 (en) * 1999-10-20 2004-03-09 Immco Diagnostics Immunological assay for detection of antibodies in celiac disease
WO2004106939A3 (fr) * 2003-06-03 2005-03-17 Scherrer Inst Paul Procede de liaison de chelateurs bifonctionnels et complexes de metal de transition (radioactif) a des proteines et des peptides
WO2005038045A1 (fr) * 2003-10-17 2005-04-28 Peoplebio, Inc. Methode et trousse pour mesurer l'activite du facteur xiii
CN115718180A (zh) * 2021-08-24 2023-02-28 黄埔海关技术中心 谷物重金属快速检测仪及其检测方法
CN119000963A (zh) * 2024-10-23 2024-11-22 浙江大学 一种基于sam与sah精确定量的cmt3蛋白活性体外检测方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4286528A1 (fr) * 2022-06-01 2023-12-06 Technische Universität Darmstadt Procédé et dispositif de détection des transglutaminases

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3834233A1 (de) * 1988-10-07 1990-04-19 Max Planck Gesellschaft Verfahren zur aktivitaetsbestimmung von transglutaminasen

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Publication number Priority date Publication date Assignee Title
JPS6035119B2 (ja) * 1982-06-11 1985-08-13 株式会社 ヤトロン 血漿中のｘ３因子を測定する方法
DE3811647A1 (de) * 1988-04-07 1989-10-26 Behringwerke Ag Verfahren und verpackung enthaltend mittel zur kinetischen bestimmung von faktor xiii

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3834233A1 (de) * 1988-10-07 1990-04-19 Max Planck Gesellschaft Verfahren zur aktivitaetsbestimmung von transglutaminasen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CLINICAL CHEMISTRY Bd. 31, Nr. 1, 1986, WINSTON-SALEM NC USA Seiten 35 - 40; L. MUSZBEK ET AL: 'Kinetic determination of blood coagulation factor XIII in plasma.' *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998058078A1 (fr) * 1997-06-18 1998-12-23 Korea Green Cross Corporation Procede permettant de determiner l'activite enzymatique du facteur xiii de coagulation sanguine a l'aide d'un substrat consistant en un monomere de fibrine purifie
US6406874B1 (en) 1997-06-18 2002-06-18 Korea Green Cross Corporation Method of determining the enzymatic activity of the blood coagulation factor XIII using purified fibrin monomer as a substrate
WO2001029090A1 (fr) * 1999-10-20 2001-04-26 Immco Diagnostics Methode de dosage immunologique servant a detecter des anticorps d'une maladie coeliaque
US6703208B1 (en) * 1999-10-20 2004-03-09 Immco Diagnostics Immunological assay for detection of antibodies in celiac disease
WO2004106939A3 (fr) * 2003-06-03 2005-03-17 Scherrer Inst Paul Procede de liaison de chelateurs bifonctionnels et complexes de metal de transition (radioactif) a des proteines et des peptides
WO2005038045A1 (fr) * 2003-10-17 2005-04-28 Peoplebio, Inc. Methode et trousse pour mesurer l'activite du facteur xiii
CN115718180A (zh) * 2021-08-24 2023-02-28 黄埔海关技术中心 谷物重金属快速检测仪及其检测方法
CN119000963A (zh) * 2024-10-23 2024-11-22 浙江大学 一种基于sam与sah精确定量的cmt3蛋白活性体外检测方法

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