CA2181875A1 - Processes for quantifying activated factors - Google Patents
Processes for quantifying activated factorsInfo
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- CA2181875A1 CA2181875A1 CA 2181875 CA2181875A CA2181875A1 CA 2181875 A1 CA2181875 A1 CA 2181875A1 CA 2181875 CA2181875 CA 2181875 CA 2181875 A CA2181875 A CA 2181875A CA 2181875 A1 CA2181875 A1 CA 2181875A1
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- Prior art keywords
- enzyme
- activated
- binding partner
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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
- G01N33/53—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
- 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
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- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to sensitive methods for the selective determination of single factors (activated coagulation proteases) for example in factor concentrates such as PPSB, i.e. in mixtures of activated and non-activated factors.
Description
- BEHRINGWERKE AKTIENGESELLSCHAFT 1995/B017 - Ma 1073 Dr.Lp/hg Processes for quantifying activated factors _ _ _ _______________________ The invention relates to sensitive processes for the selective determination of single factors (activated coagulation proteases) for example in factor concentrates such as PPSB, i.e. in mixtures of activated and non-activated factors.
Congenital and acquired deficiencies of plasma proteins or blood cells are currently substituted by infusing plasma, cell concentrates as well as proteins and factor concentrates which have been purified by plasma pool~. In addition to the potential hazard of virus transmissions, which are m;n;m; zed by single-donor testing and suitable virus reduction and inactivation processes, undesirable reactions, such as, for example, drop in blood pressure, and thrombogenic or hemagogic effects, are observed. These side effects can be caused by the presence of so-called activated factors. Even though these products are purified under the mildest possible conditions and are subject to suitable quality checks and licensing criteria, the abovementioned side effects have been reported in individual cases.
Retrospectively, analyses have frequently confirmed the suspected presence of activated factors in these products.
The concentrates prepared from blood plasma can contain traces of other plasma proteins which usually do not noticeably affect efficacy and safety of the product.
If these proteins, i.e. enzymes/proteases, are present in the activated state, this may have fatal consequences for the patient post-application. In addition to the activated form of prothrombin, i.e.
thrombin, traces of the activated coagulation factors VII, IX, X, XI, XII or of prekallikrein or of protein C
(F VIIa, F IXa, F Xa, F XIa, F XIIa, kallikrein, APC) or of the complement system may also be present.
~owever, even the purified proteins themselves, whether they have been prepared by conventional precipitation and chromatographic methods from plasma or other starting materials or by me~n~ of recombinant technology, may be partially activated. Of equal importance is the detection of these activated factors.
The fact that these are already active in minute concentrations and may cause triggering or reinforcing in-vivo effects requires tests which are as sensitive and specific as possible.
Currently used methods record for example the enzyma-tic/proteolytic activities of these factors, while their zymogens are, accordingly, inactive. It is conventionally attempted to record these activities with the aid of chromogenic substrates, the amidolytic liberation of, for example~ p-nitrophenylanilide or fluorogenic groups being recorded and evaluated photometrically. Even though these substrates are optimized for specific proteases or activated factors, they are also cleaved by other proteases to a lesser or greater extent. This is why differentiation and quantification of single components - and therefore also more or less "harmful" activities - is particularly difficult in complex protein (protease) mixtures such as, for example, prothrombin complex concentrates. It is only made possible when a comparison measurement i8 carried out in the presence of a highly specific inhibitor, as in the case of thrombin +/- hirudin, with which the specific activity can be eliminated. In the case of the other proteases, this has not been possible to date since the inhibitors available are not sufficiently specific, albeit highly potent in some cases.
Immunochemical detection systems such as, for example, RIA, E~ISA etc. are highly selective and sensitive methods. Such test systems, which could specifically `21~1875 _ quantify the activated protease, would~ be suitable.
However, obtaining antibodies which, on~the one hand, exclusively recognize the activated form of a factor is frequently difficult or, in many cases,` has not been carried out successfully to date. However, this is required for the products described, since a mixture of zymogen and activated form is present. On the other hand, protease inhibitor complexes which are present must not be measured simultaneously since they would falsify the results of a quantification of the free, blologically active form. These complexes can be formed during protein purification or by deliberately added inhibitors. Accordingly, the aim ia to provide suitable assay systems to quantify these active factoræ/proteaæeæ, in particular the factors VIIa, F
IXa, F Xa, F XIa, F XIIa, kallikrein and APC as well as proteases of the complement system. A homogeneous assay method in the liquid phase has been described for free thrombin with the aid of the thrombin/antithrombin III
complex in coagulation products (Kohler et al., Thromb.
Res. 1990; 60: 63-70).
In the present invention, the product or concentrate is treated with an enzyme/protease binding partner, pre-ferably an inhibitor, which can be bound by anantibody, the mixture is incubated as required, and the enzyme-/protease inhibitor complexes generated are quantified by means of solid-phase ELISA (enzyme-linked ;mmllnosorbent assay).
Suitable protease inhibitors are known binding partners, such as antithrombin III (AT III), a2-macroglobulin, al-antitrypsin, a2-antiplasmin, inter-a-trypsin inhibitor, Cl-inhibitor, protein C inhibitor and the like, preferably AT III, optionally in the presence of heparin, and al-antitrypsin. AT III is the most important physiological regulator/inhibitor of the coagulation system which reacts with activated factors with a different affinity/rate and forms a complex.
2181~75 While, for example, the reaction with thrombin proceeds relatively rapidly and is accelerated greatly by heparin, as is well known, an interaction with, for example, F VIIa proceedæ merely very slowly at room temperature or 37C or only when F VIIa is present as a complex with thromboplastin. Another possibility was described by Godal et al. (Thromb. Res. 1974; 5: 773-775), where F VIIa is incubated with ATIII/heparin for several hours under cold conditions and the remaining coagulation-promoting F VIIa is subsequently determined in a suitable activity assay.
In contrast, specificity and sensitivity are increased when the protease inhibitor complexes formed are quan-tified with the aid of an ;mmllnological assay. Accord-ingly, polyclonal (Pab) or monoclonal (Mab) antibodies or their F(ab') or F(ab) fragments, preferably a monoclorlal ab or its Fab, are coupled to, or adsorbed onto, the solid phase by known methodq, the st~n~rd or sample solution is incubated therewith, the solid phase is washed, and the bound complexes are incubated with a polyclonal or monoclonal ab, or Fab against the compiex partner (protease or inhibitor). This ab is conjugated for example with an enzyme, such as POD. After a suitable enzyme substrate has been added, the substrate conversion rate is measured and the concentration of the bound complex is determined by means of a standard curve. The concentration of the protease which exists in free form in the product can be calculated accordingly.
If a limited amount of protease inhibitor complexes (for example for preparation reasons) exists already prior to the addition of an adequately high concentration of the inhibitor (fully to convert the free protease into complexes), then the control value (product dilution without additionally added inhibitor) is subtracted from the sample value whereby the concentration of the free, activated protease is calculated.
2 ! 8 1 8 75 -In a preferred embodiment (A), known methods are used for directly coupling the inhibitor to the solid phase or binding it by means of an antibody (to the inhibitor) which is coupled to the solid phase, and thus exposed to the free proteases in solution. After incubation and formation of the now immobilized inhibitor protease complexes, the solid phase is washed and the complexes are detected by means of a suitable (conjugated) antibody to the prot~ase or to a neoepitope of the complex (or by means of a conjugated antibody to these antibodies).
If appropriate, the detecting antibody/Fab may already be pre ent while the solid phase i~ incubated with the sample solution.
In a further preferred ~mhodiment (B), an antibody/Fab suitable for the enzyme/protease to be detected is bound to the solid phase (or an antibody to this antibody, for example goat-anti-mouse, which presents the specific antibody). The solution which comprises the free enzyme/protease is brought into contact with the solid phase and incubated. After the free, activated protease (as well as non-activated enzymes/proteases in solution) have bound, the solid phase is washed and incubated with a solution comprising the corresponding inhibitor. After the solid phase has been washed, the complexes are detected with the aid of a conjugated antibody which binds to the inhibitor or to a neoepitope of the complex. Equally, the solution which comprises the activated, free protease may be treated with the inhibitor before (and incubation) or during the incubation with the contact phase.
If appropriate, the detecting antibody/Fab may already be present during incubation of the solid phase with the inhibitor.
The abovementioned embodiments are used depending on the intended aim: while, in embodiment (A), the -immobilized binding partner (here the inhibitor) binds exclusively activated proteases, allowing traces of these non-complex molecules to be detected, embodiment (B) `additionally creates the possibility for the limited enzyme/protease inhibitor complex which may be present to be measured even before the inhibitor is added. A parallel ~Y~m; n~ tion of the sample solution (+/- inhibitor) followed by subtraction ((+) inhibitor minus (-) inhibitor) allows both pieces of information to be obtained.
Preferred combinations of binding partners for determining activated factorR are AT III, if appropriate, F VII a, F IX a, F X a, plus heparin: F XI a, F XII a C1 inactivator: F XI a, kallikrein, Clr a1 antitrypsin: activated protein C, elastase, other serine proteases Protein C inhibitor: activated protein C
Example:
Quantification of FIXa-AT III complex (cx) Materials:
Microtiter plates were coated with monoclonal antibodies (Mab, 10 ~g/ml) to FIX/FIXa and washed.
Peroxidase (POD)-conjugated Mab to ATIII were used to detect FIXa-ATIII-cx.
AT III (Kybernin~), Mab to FIX and to ATIII were obtained from Behringwerke AG (Germany). Heparin (Liquemin~) was obtained from Hoffmann-La-Roche GmbH
(Germany). Factors IX and IXa were acquired from _, r Calbiochem (USA) and Serbio (France). The chromogenic ~~
substrate S-2765 was from Chromogenix AB (Sweden).
Preparation of an FIXa-ATIII-cx st~n~rd:
500 ~l of a buffer solution comprising FIXa (2~g/ml) were incubated with an equal volume of an ATIII/heparin solution comprising a 10-fold molar excess (dilution from 10 I.U./ml of ATIII/20 I.U./ml of heparin) for 90 minutes at room temperature (RT). The complete com-plexing of FIXa with ATIII was monitored by photometric determination of the amidolytic FIXa-activity by means of S-2765 in comparison with the control batch (without ATIII). After the abovementioned incubation time, activity in the batch comprising FIXa/ATIII/heparin was no longer detectable - FIXa had been converted fully into the FIXa-ATIII-cx.
Establishing an FIXa-ATIII-cx standard curve:
Aliquots of the FIXa-ATIII-cx standard solution (1.0 ~ g/ml) were diluted stepwise down to a factor of 1250 and the dilutions used for establishing a standard curve. In each case 50 ~l of a sample buffer were introduced into the wells of the microtiter plates coated with anti-FIX-Mab, and in each case 50 ~l of the cx solutions were added.
After incubation for 1 hour at 37C, the solid phase was washed three times, and in each case 100 ~l of the solution comprising anti-ATIII-Mab-POD were added.
After reincubation for 1 hour at 37C, the plates were washed three times. In each case 100 ~l of POD-substrate solution (O-phenylenediamine hydrochloride) were pipetted into each well, the plates were incubated for 20 minutes at RT (in the dark), the reaction was stopped, and the OD4g2 was measured:
21&1875 FIXa-ATIII-cx (ng/ml) OD4g2 (mean value of - duplicate tests) 0.8 0.027 1.6 0.028 5-3.1 0.043 6.25 0.09 12.5 0.13 0.24 0.42 10100 0.71 125 0.86 250 1.34 500 1.98 Accordingly, this gave a linear measurement range of at least 1.5 to 150 ng/ml (when plotting log concentration cx versus log OD4g2)-Quantification of FIXa-ATIII-cx in FIX/FIXa mixtures:
The following mixtures of FIX, FIXa and ATIII/heparin were prepared, incubated for 90 minutes at RT and treated as sample as described above:
End concentration in the batch 1. 40 ~l FIX (50 ng/ml) 20 ng/ml 40 ~l FIXa (150 ng/ml) 60 ng/ml 20 ~l ATIII/Hep (5 U/ml) 1 U/ml 2. 40 ~l FIX (100 ng/ml) 40 ng/ml 40 ~l FIXa (50 ng/ml) 20 ng/ml 20 ~l ATIII/Hep (5 U/ml) 1 U/ml 3. 40 ~1 FIX (450 ng/ml) 180 ng/ml 40 ~l FIXa (50 ng/ml) 20 ng/ml 20 ~l ATIII/Hep (5 U/ml) 1 U/ml Control batches comprising the factors IX, IXa and ATIII/Hep in each case separately were also prepared at the concentrations shown from 1. to 3.. Neither FIX, -g nor IXa nor ATIII/Hep showed a reaction in this assay system. This equally applied to the mixture FIX/FIXa (without subsequent incubation with ATIII/Hep, which guaranteed selectivity for the FIXa-ATIII-cx.
FIXa-ATIII-cx formed was quantified using the above-mentioned standard curve. The table shows the mean values of the results of test dilutions.
Batch No. FIXa-ATIII-cx Recovery rate Dilutions (factor of (ng/ml) (%) x) 1 58.6 98 2/4/8/16/32
Congenital and acquired deficiencies of plasma proteins or blood cells are currently substituted by infusing plasma, cell concentrates as well as proteins and factor concentrates which have been purified by plasma pool~. In addition to the potential hazard of virus transmissions, which are m;n;m; zed by single-donor testing and suitable virus reduction and inactivation processes, undesirable reactions, such as, for example, drop in blood pressure, and thrombogenic or hemagogic effects, are observed. These side effects can be caused by the presence of so-called activated factors. Even though these products are purified under the mildest possible conditions and are subject to suitable quality checks and licensing criteria, the abovementioned side effects have been reported in individual cases.
Retrospectively, analyses have frequently confirmed the suspected presence of activated factors in these products.
The concentrates prepared from blood plasma can contain traces of other plasma proteins which usually do not noticeably affect efficacy and safety of the product.
If these proteins, i.e. enzymes/proteases, are present in the activated state, this may have fatal consequences for the patient post-application. In addition to the activated form of prothrombin, i.e.
thrombin, traces of the activated coagulation factors VII, IX, X, XI, XII or of prekallikrein or of protein C
(F VIIa, F IXa, F Xa, F XIa, F XIIa, kallikrein, APC) or of the complement system may also be present.
~owever, even the purified proteins themselves, whether they have been prepared by conventional precipitation and chromatographic methods from plasma or other starting materials or by me~n~ of recombinant technology, may be partially activated. Of equal importance is the detection of these activated factors.
The fact that these are already active in minute concentrations and may cause triggering or reinforcing in-vivo effects requires tests which are as sensitive and specific as possible.
Currently used methods record for example the enzyma-tic/proteolytic activities of these factors, while their zymogens are, accordingly, inactive. It is conventionally attempted to record these activities with the aid of chromogenic substrates, the amidolytic liberation of, for example~ p-nitrophenylanilide or fluorogenic groups being recorded and evaluated photometrically. Even though these substrates are optimized for specific proteases or activated factors, they are also cleaved by other proteases to a lesser or greater extent. This is why differentiation and quantification of single components - and therefore also more or less "harmful" activities - is particularly difficult in complex protein (protease) mixtures such as, for example, prothrombin complex concentrates. It is only made possible when a comparison measurement i8 carried out in the presence of a highly specific inhibitor, as in the case of thrombin +/- hirudin, with which the specific activity can be eliminated. In the case of the other proteases, this has not been possible to date since the inhibitors available are not sufficiently specific, albeit highly potent in some cases.
Immunochemical detection systems such as, for example, RIA, E~ISA etc. are highly selective and sensitive methods. Such test systems, which could specifically `21~1875 _ quantify the activated protease, would~ be suitable.
However, obtaining antibodies which, on~the one hand, exclusively recognize the activated form of a factor is frequently difficult or, in many cases,` has not been carried out successfully to date. However, this is required for the products described, since a mixture of zymogen and activated form is present. On the other hand, protease inhibitor complexes which are present must not be measured simultaneously since they would falsify the results of a quantification of the free, blologically active form. These complexes can be formed during protein purification or by deliberately added inhibitors. Accordingly, the aim ia to provide suitable assay systems to quantify these active factoræ/proteaæeæ, in particular the factors VIIa, F
IXa, F Xa, F XIa, F XIIa, kallikrein and APC as well as proteases of the complement system. A homogeneous assay method in the liquid phase has been described for free thrombin with the aid of the thrombin/antithrombin III
complex in coagulation products (Kohler et al., Thromb.
Res. 1990; 60: 63-70).
In the present invention, the product or concentrate is treated with an enzyme/protease binding partner, pre-ferably an inhibitor, which can be bound by anantibody, the mixture is incubated as required, and the enzyme-/protease inhibitor complexes generated are quantified by means of solid-phase ELISA (enzyme-linked ;mmllnosorbent assay).
Suitable protease inhibitors are known binding partners, such as antithrombin III (AT III), a2-macroglobulin, al-antitrypsin, a2-antiplasmin, inter-a-trypsin inhibitor, Cl-inhibitor, protein C inhibitor and the like, preferably AT III, optionally in the presence of heparin, and al-antitrypsin. AT III is the most important physiological regulator/inhibitor of the coagulation system which reacts with activated factors with a different affinity/rate and forms a complex.
2181~75 While, for example, the reaction with thrombin proceeds relatively rapidly and is accelerated greatly by heparin, as is well known, an interaction with, for example, F VIIa proceedæ merely very slowly at room temperature or 37C or only when F VIIa is present as a complex with thromboplastin. Another possibility was described by Godal et al. (Thromb. Res. 1974; 5: 773-775), where F VIIa is incubated with ATIII/heparin for several hours under cold conditions and the remaining coagulation-promoting F VIIa is subsequently determined in a suitable activity assay.
In contrast, specificity and sensitivity are increased when the protease inhibitor complexes formed are quan-tified with the aid of an ;mmllnological assay. Accord-ingly, polyclonal (Pab) or monoclonal (Mab) antibodies or their F(ab') or F(ab) fragments, preferably a monoclorlal ab or its Fab, are coupled to, or adsorbed onto, the solid phase by known methodq, the st~n~rd or sample solution is incubated therewith, the solid phase is washed, and the bound complexes are incubated with a polyclonal or monoclonal ab, or Fab against the compiex partner (protease or inhibitor). This ab is conjugated for example with an enzyme, such as POD. After a suitable enzyme substrate has been added, the substrate conversion rate is measured and the concentration of the bound complex is determined by means of a standard curve. The concentration of the protease which exists in free form in the product can be calculated accordingly.
If a limited amount of protease inhibitor complexes (for example for preparation reasons) exists already prior to the addition of an adequately high concentration of the inhibitor (fully to convert the free protease into complexes), then the control value (product dilution without additionally added inhibitor) is subtracted from the sample value whereby the concentration of the free, activated protease is calculated.
2 ! 8 1 8 75 -In a preferred embodiment (A), known methods are used for directly coupling the inhibitor to the solid phase or binding it by means of an antibody (to the inhibitor) which is coupled to the solid phase, and thus exposed to the free proteases in solution. After incubation and formation of the now immobilized inhibitor protease complexes, the solid phase is washed and the complexes are detected by means of a suitable (conjugated) antibody to the prot~ase or to a neoepitope of the complex (or by means of a conjugated antibody to these antibodies).
If appropriate, the detecting antibody/Fab may already be pre ent while the solid phase i~ incubated with the sample solution.
In a further preferred ~mhodiment (B), an antibody/Fab suitable for the enzyme/protease to be detected is bound to the solid phase (or an antibody to this antibody, for example goat-anti-mouse, which presents the specific antibody). The solution which comprises the free enzyme/protease is brought into contact with the solid phase and incubated. After the free, activated protease (as well as non-activated enzymes/proteases in solution) have bound, the solid phase is washed and incubated with a solution comprising the corresponding inhibitor. After the solid phase has been washed, the complexes are detected with the aid of a conjugated antibody which binds to the inhibitor or to a neoepitope of the complex. Equally, the solution which comprises the activated, free protease may be treated with the inhibitor before (and incubation) or during the incubation with the contact phase.
If appropriate, the detecting antibody/Fab may already be present during incubation of the solid phase with the inhibitor.
The abovementioned embodiments are used depending on the intended aim: while, in embodiment (A), the -immobilized binding partner (here the inhibitor) binds exclusively activated proteases, allowing traces of these non-complex molecules to be detected, embodiment (B) `additionally creates the possibility for the limited enzyme/protease inhibitor complex which may be present to be measured even before the inhibitor is added. A parallel ~Y~m; n~ tion of the sample solution (+/- inhibitor) followed by subtraction ((+) inhibitor minus (-) inhibitor) allows both pieces of information to be obtained.
Preferred combinations of binding partners for determining activated factorR are AT III, if appropriate, F VII a, F IX a, F X a, plus heparin: F XI a, F XII a C1 inactivator: F XI a, kallikrein, Clr a1 antitrypsin: activated protein C, elastase, other serine proteases Protein C inhibitor: activated protein C
Example:
Quantification of FIXa-AT III complex (cx) Materials:
Microtiter plates were coated with monoclonal antibodies (Mab, 10 ~g/ml) to FIX/FIXa and washed.
Peroxidase (POD)-conjugated Mab to ATIII were used to detect FIXa-ATIII-cx.
AT III (Kybernin~), Mab to FIX and to ATIII were obtained from Behringwerke AG (Germany). Heparin (Liquemin~) was obtained from Hoffmann-La-Roche GmbH
(Germany). Factors IX and IXa were acquired from _, r Calbiochem (USA) and Serbio (France). The chromogenic ~~
substrate S-2765 was from Chromogenix AB (Sweden).
Preparation of an FIXa-ATIII-cx st~n~rd:
500 ~l of a buffer solution comprising FIXa (2~g/ml) were incubated with an equal volume of an ATIII/heparin solution comprising a 10-fold molar excess (dilution from 10 I.U./ml of ATIII/20 I.U./ml of heparin) for 90 minutes at room temperature (RT). The complete com-plexing of FIXa with ATIII was monitored by photometric determination of the amidolytic FIXa-activity by means of S-2765 in comparison with the control batch (without ATIII). After the abovementioned incubation time, activity in the batch comprising FIXa/ATIII/heparin was no longer detectable - FIXa had been converted fully into the FIXa-ATIII-cx.
Establishing an FIXa-ATIII-cx standard curve:
Aliquots of the FIXa-ATIII-cx standard solution (1.0 ~ g/ml) were diluted stepwise down to a factor of 1250 and the dilutions used for establishing a standard curve. In each case 50 ~l of a sample buffer were introduced into the wells of the microtiter plates coated with anti-FIX-Mab, and in each case 50 ~l of the cx solutions were added.
After incubation for 1 hour at 37C, the solid phase was washed three times, and in each case 100 ~l of the solution comprising anti-ATIII-Mab-POD were added.
After reincubation for 1 hour at 37C, the plates were washed three times. In each case 100 ~l of POD-substrate solution (O-phenylenediamine hydrochloride) were pipetted into each well, the plates were incubated for 20 minutes at RT (in the dark), the reaction was stopped, and the OD4g2 was measured:
21&1875 FIXa-ATIII-cx (ng/ml) OD4g2 (mean value of - duplicate tests) 0.8 0.027 1.6 0.028 5-3.1 0.043 6.25 0.09 12.5 0.13 0.24 0.42 10100 0.71 125 0.86 250 1.34 500 1.98 Accordingly, this gave a linear measurement range of at least 1.5 to 150 ng/ml (when plotting log concentration cx versus log OD4g2)-Quantification of FIXa-ATIII-cx in FIX/FIXa mixtures:
The following mixtures of FIX, FIXa and ATIII/heparin were prepared, incubated for 90 minutes at RT and treated as sample as described above:
End concentration in the batch 1. 40 ~l FIX (50 ng/ml) 20 ng/ml 40 ~l FIXa (150 ng/ml) 60 ng/ml 20 ~l ATIII/Hep (5 U/ml) 1 U/ml 2. 40 ~l FIX (100 ng/ml) 40 ng/ml 40 ~l FIXa (50 ng/ml) 20 ng/ml 20 ~l ATIII/Hep (5 U/ml) 1 U/ml 3. 40 ~1 FIX (450 ng/ml) 180 ng/ml 40 ~l FIXa (50 ng/ml) 20 ng/ml 20 ~l ATIII/Hep (5 U/ml) 1 U/ml Control batches comprising the factors IX, IXa and ATIII/Hep in each case separately were also prepared at the concentrations shown from 1. to 3.. Neither FIX, -g nor IXa nor ATIII/Hep showed a reaction in this assay system. This equally applied to the mixture FIX/FIXa (without subsequent incubation with ATIII/Hep, which guaranteed selectivity for the FIXa-ATIII-cx.
FIXa-ATIII-cx formed was quantified using the above-mentioned standard curve. The table shows the mean values of the results of test dilutions.
Batch No. FIXa-ATIII-cx Recovery rate Dilutions (factor of (ng/ml) (%) x) 1 58.6 98 2/4/8/16/32
Claims (9)
1. A heterogeneous immunochemical method of detecting and determining activated enzymes in a sample, which method embraces the following steps:
a) incubation of the sample with a first binding partner which is specific for the activated state of the enzyme, and b) detection of the formation of the enzyme/binding partner complex by reacting the enzyme/binding partner complex with a second specific binding partner which specifically reacts with the first binding partner which is specific for the activated state of the enzyme, the second specific binding partner being directly or indirectly provided with a signal-producing labeling.
a) incubation of the sample with a first binding partner which is specific for the activated state of the enzyme, and b) detection of the formation of the enzyme/binding partner complex by reacting the enzyme/binding partner complex with a second specific binding partner which specifically reacts with the first binding partner which is specific for the activated state of the enzyme, the second specific binding partner being directly or indirectly provided with a signal-producing labeling.
2. A method as claimed in claim 1, wherein the enzyme/binding partner complex is immobilized.
3. A method as claimed in claim 2, wherein immobiliz-ation is effected by the first specific binding partner, which is bound to a solid phase.
4. A method as claimed in claim 3, wherein the immo-bilized enzyme/binding partner complex is detected by means of an antibody which is specific for the enzyme component or for the enzyme/binding partner complex, this antibody being directly or indirectly provided with a signal-producing labeling.
5. A method as claimed in claim 1, wherein the acti-vated enzyme is immobilized by an enzyme-specific antibody bound to a solid phase, and the immobilized activated enzyme is incubated with a binding partner specific for the activated state of the enzyme, this binding partner being directly or indirectly provided with a signal-producing labeling.
6. A method as claimed in any of claims 1 to 5, wherein the activated enzyme is an activated coagulation factor.
7. A method as claimed in any of claims 1 to 6, wherein the signal-producing labeling is a chemiluminescent or fluorogenic labeling.
8. A method as claimed in any of claims 1 to 7, wherein the signal-producing labeling is an enzyme, preferably horseradish peroxidase.
9. The use of a method as claimed in any of claims 1 to 8 for determining activated coagulation factors in mixtures of activated and non-activated factors.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19526700 | 1995-07-24 | ||
| DE19526700.1 | 1995-07-24 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2181875A1 true CA2181875A1 (en) | 1997-01-25 |
Family
ID=7767450
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2181875 Abandoned CA2181875A1 (en) | 1995-07-24 | 1996-07-23 | Processes for quantifying activated factors |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0759556A3 (en) |
| JP (1) | JPH0933523A (en) |
| AU (1) | AU714952B2 (en) |
| CA (1) | CA2181875A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2970417A1 (en) * | 2011-01-19 | 2012-07-20 | Lfb Biotechnologies | Kit, useful as a drug for treating severe sepsis or septic shock, comprises protein C and alpha-1-antitrypsin |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3016575A1 (en) * | 1980-04-30 | 1981-11-05 | Merck Patent Gmbh, 6100 Darmstadt | METHOD FOR THE IMMUNOLOGICAL DETERMINATION OF ENZYMES, MEANS FOR CARRYING OUT THE METHOD AND ITS RELATIONSHIP |
| DE3172143D1 (en) * | 1980-09-30 | 1985-10-10 | Cornell Res Foundation Inc | Process for determining inhibitor-enzyme complexes |
| US4849353A (en) * | 1980-09-30 | 1989-07-18 | Cornell Research Foundation, Inc. | Immunocapture of enzyme inhibitor, enzyme complexes and uses thereof |
| NL8802710A (en) * | 1988-11-04 | 1990-06-01 | Tno | METHOD FOR DETERMINING AN ENZYME AND SUITABLE KIT AND SUBSTANCE. |
| GB8901859D0 (en) * | 1989-01-27 | 1989-03-15 | Shield Diagnostics Limited | Diagnostic test |
| AU628960B2 (en) * | 1989-04-07 | 1992-09-24 | Teijin Limited | Method of immunologically assaying human thrombin- antithrombin III complex, assay reagent and kit therefor |
| GB9119102D0 (en) * | 1991-09-07 | 1991-10-23 | Biosyn Ltd | Bioimmunassay |
-
1996
- 1996-06-25 EP EP96110212A patent/EP0759556A3/en not_active Withdrawn
- 1996-07-22 AU AU60615/96A patent/AU714952B2/en not_active Ceased
- 1996-07-23 JP JP19263896A patent/JPH0933523A/en active Pending
- 1996-07-23 CA CA 2181875 patent/CA2181875A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU6061596A (en) | 1997-01-30 |
| EP0759556A3 (en) | 1998-05-20 |
| AU714952B2 (en) | 2000-01-13 |
| JPH0933523A (en) | 1997-02-07 |
| EP0759556A2 (en) | 1997-02-26 |
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