WO1993005397A1 - Immunoassay for proteases - Google Patents
Immunoassay for proteases Download PDFInfo
- Publication number
- WO1993005397A1 WO1993005397A1 PCT/GB1992/001626 GB9201626W WO9305397A1 WO 1993005397 A1 WO1993005397 A1 WO 1993005397A1 GB 9201626 W GB9201626 W GB 9201626W WO 9305397 A1 WO9305397 A1 WO 9305397A1
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- WO
- WIPO (PCT)
- Prior art keywords
- assay according
- protease
- peptide
- enzyme
- inhibitor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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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
Definitions
- This invention relates to a novel solid phase Immunoassay for proteases.
- Psychrotrophic bacteria present in raw milk can increase in number during refrigerated storage and produce heat-stable proteases which survive pasteurisation and ultra high temperature (UHT) treatment. These enzymes can biochemically alter milk eventually causing spoilage. As a result, the processing properties of the milk can be adversely affected and the quality of products made from the milk impaired. It is commercially desirable to be able to test in advance of any processing whether the milk contains these enzymes. It is known to detect proteases by substrate or bioimmunoassay methods, assays which require long incubation times. In the known methods the enzymes can be inactivated by endogenous proteinaceous inhibitors in the milk being tested so that an incorrect result is obtained from the assay. There is a need for a test for incipient bacterial spoilage in milk which can be carried out quickly by unskilled operatives and which gives accurate results, whereby poor quality milk and possibly also infected herds can be identified.
- the invention provides a solid phase immunoassay method for detecting a protease in a material, comprising contacting the material under test with a peptide inhibitor for the protease to be detected, said inhibitor being immobilised on a solid phase, whereby the enzyme in the material becomes bound to the solid phase and detecting the bound enzyme with a tracer for the enzyme.
- the assay of the invention may be carried out on any sample which Is suspected of containing one or more than one protease and may find use in both clinical and non-clinical situations.
- samples may be biological samples such as blood, urine, serum, plasma, tumour homogenates and milk.
- the assay of the invention finds particular use in the detection of proteases, especially serine proteases and particularly plasmin in milk.
- Bovine mastitis inflammation of the mammary gland, is the most common and most costly disease that afflicts dairy cows world-wide.
- mastitis blood proteins are passively transferred Into the milk as a result of Inflammation.
- plasmin a trypsin-like serine protease
- Plasmin has been implicated in the hydrolysis of caseins during Incubation or storage of good quality milk, thereby affecting the quality of dairy products produced.
- proteases The peptide inhibitor for the target protease is chosen having regard to the target enzyme that is to be detected.
- Proteases can be subdivided into general groups depending on their mode of action. Proteases may be described as being trypsin-like, elastase-like or chymotrypsin-like. Trypsin, elastase and chymotrypsin are proteases which have been well-characterised and this description of proteases is well understood in the art. The function of proteases is to fragment a protein by hydrolysing the peptide bonds of that protein.
- Trypsin and trypsin-like proteases hydrolyse peptide bonds between either the C-terminal of the ami no acid residues lysine or arginine and the N-terminal of any other amino acid residue.
- Chymotrypsin and chymotrypsin-like enzymes hydrolyse the peptide bond between the C-terminal of either phenylalanine, tryptophan or tyrosine and the N-terminal of any other amino acid.
- Elastase and elastase-like enzymes hydrolyse the peptide bond between the C-terminal of valine, alanine or norleucine and the N-terminal of any other amino acid.
- the protease inhibitor is preferably an irreversible inhibitor.
- Proteases may be inhibited by peptides, some of which are known.
- the peptide inhibitor of the target enzyme for use in the assay of the invention is a synthetic peptide of 3-20 amino acid residues in length, more preferably of between 3 and 10 amino acid residues and even more preferably 3 and 5 amino acid residues in length.
- the amino add residues may be naturally occurring amino acids or unnatural amino adds and the Inhibitor is made irreversible by the addition to the C-terminal of the peptide of a chemical group R which is a group capable of modifying the peptide irreversibly, e.g. chloromethyIketone, diphenylphosphorate or fluoromethylketone.
- the amino acid residue Immediately preceding the R chemical group i.e. the C-terminal amino acid residue of the peptide is a residue "Y" which is the amino acid residue normally recognised by the target protease as the signal for hydrolysis.
- the Y residue is the amino acid residue normally recognised by the target protease as the signal for hydrolysis.
- the Y residue is preferably tyrosine, tryptophan or phenylalanine.
- the Y residue is preferably valine, alanine or norleucine.
- the tracer for the bound target enzyme is preferably a labelled ligand which will usually be a specific binding ligand such as an antibody.
- a labelled ligand which will usually be a specific binding ligand such as an antibody.
- Any known method of labelling enzymes for assay purposes may be adopted, e.g. with a detectable marker for example another enzyme, radioisotope, spin label, fluorescent material or the like.
- Particularly preferred tracers are polyclonal and monoclonal antibodies raised against the target enzyme or the target enzyme inactivated with the specific inhibitor, with an enzyme label conjugated directly on to the primary antibody or to a secondary antibody directed against the host IgG of an animal in which the primary antibody was raised.
- the solid phase can take any suitable form known in heterogeneous assays, including plates, particles, strips, rods, dipsticks, membranes, microtitre wells and so on.
- the target enzyme Once the target enzyme has been immobilised on the solid phase it can be brought into contact with the tracer, for example a solution of the labelled antibody.
- the method of the invention can be used to give a very rapid qualitative analysis of the enzyme under investigation or a quantitative determination. For the latter, mlcrotitre plate reading technology can be applied.
- ImM glutaric anhydride 200 ⁇ l/well of ImM glutaric anhydride was coated onto Nunc Covalink NH microtitre plates overnight at 4°C. The plates were washed twice with distilled water and coated with 100 ⁇ l/well of ImM 1-ethyl-3 (-3 dimethylaminopropyl) carbodiimide and 100 ⁇ l/well of 90 ⁇ M D-Phenylalanyl-L-phenylalanyl-L-arginine chloromethylketone overnight at 4oC.
- the plates were washed twice with PBST and 100 ⁇ l/well of a bacterial protease diluted 1/10 in bovine milk was incubated overnight at 4°C.
- the plates were washed twice and pre-immune or anti-protease polyclonal antisera were added 100 ⁇ l/well at a dilution of 1/35 for 1 hour at 37°C. Following five washes, 100 ⁇ l of goat anti-rabbit Ig conjugated to horse-radish
- HRPO peroxidase
- the invention is not restricted to the detection of proteases in milk. It can be used for the detection of enzymes in other complex fluids such as coagulation proteases in blood and tumour specific proteolytic activity. This is outlined in the
- COSTAR EIA plates for covalent binding were coated with 100 ⁇ l of 1 mM 1-ethyl-3 (-3 dimethylaminopropyl) carbodiimide and 100 ⁇ l of peptide inhibitor (see details in Table 2 below) both in distilled water, pH 6.0, overnight at 4°C. Following washing, with acidified water, the remaining reactive COOH sites were 'capped' using 300 ⁇ l of 27. Glycine methyl ester in distilled water, pH 4.5, for 3 hours at 37oC. The remaining binding sites on the plates were blocked by the addition of 300 ⁇ l of a 11 protein solution (bovine serum albumin, ovalbumin or dried milk powder) in distilled water, pH 4.5. Samples containing target enzyme or standards (100 ⁇ l) were added and incubated for 3 hours at 37°C.
- a 11 protein solution bovine serum albumin, ovalbumin or dried milk powder
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- Engineering & Computer Science (AREA)
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- Hematology (AREA)
- Urology & Nephrology (AREA)
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- Cell Biology (AREA)
- Food Science & Technology (AREA)
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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
A solid phase immunoassay method for detecting a protease in a material comprising contacting the material under test with a peptide inhibitor for the protease to be detected, said inhibitor being immobilised on a solid phase, whereby the enzyme in the material becomes bound to the solid phase and detecting the bound enzyme with a tracer for the enzyme.
Description
IMMUNOASSAY FOR PROTEASES
BACKGROUND TO THE INVENTION
Field of the Invention
This invention relates to a novel solid phase Immunoassay for proteases.
DESCRIPTION OF THE RELATED ART
Psychrotrophic bacteria present in raw milk can increase in number during refrigerated storage and produce heat-stable proteases which survive pasteurisation and ultra high temperature (UHT) treatment. These enzymes can biochemically alter milk eventually causing spoilage. As a result, the processing properties of the milk can be adversely affected and the quality of products made from the milk impaired. It is commercially desirable to be able to test in advance of any processing whether the milk contains these enzymes. It is known to detect proteases by substrate or bioimmunoassay methods, assays which require long incubation times. In the known methods the enzymes can be inactivated by endogenous proteinaceous inhibitors in the milk being tested so that an incorrect result is obtained from the assay. There is a need for a test for incipient bacterial spoilage in milk which can be carried out quickly by unskilled operatives and which gives accurate results, whereby poor quality milk and possibly also infected herds can be identified.
In addition, in clinical situations, the detection of aberrant proteolytic activity associated with pathological conditions is invaluable in diagnosis. The specificities of proteases involved in disease are well documented, for example see Kettner, C. and Shaw, E.; (1978), Biochemistry, 17, 4778-4784, Kettner, C. and Shaw, E., (1979), Thrombosis Research, 14, 969-973 and Silverberg, M. and Kaplan, A. P., (1982) Blood 60, 64-70. There are several tests which exist for enzyme detection in clinical fluids such as plasma and serum. These tests depend on the ability of proteases to cleave low molecular weight peptide substrates that bear either fluorescent or
spectrophotometric leaving groups. The single biggest drawback of these assays is that they can only be used to detect activity in plasma or serum since red blood corpuscles and platelets in more complex samples interfere with the development of colour and the fluorescence generated by the cleavage of the substrates.
SUMMARY OF THE INVENTION
Accordingly the invention provides a solid phase immunoassay method for detecting a protease in a material, comprising contacting the material under test with a peptide inhibitor for the protease to be detected, said inhibitor being immobilised on a solid phase, whereby the enzyme in the material becomes bound to the solid phase and detecting the bound enzyme with a tracer for the enzyme.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The assay of the invention may be carried out on any sample which Is suspected of containing one or more than one protease and may find use in both clinical and non-clinical situations. These samples may be biological samples such as blood, urine, serum, plasma, tumour homogenates and milk.
For example, coronary and thrombotic disease is one of the major causes of death in the western world. Irrespective of the underlying physiological causes of these illnesses, the observed clinical manifestations are a direct result of the uncontrolled activation of the proteolytic clotting cascade in blood. The ability to monitor the levels of these proteases, for example, thrombin and Factor Xa, in individuals in high risk groups would offer tremendous diagnostic potential before they presented with the clinical symptoms associated with, for example, infarction or deep vein thrombosis. This is therefore one example of a clinical situation wherein the assay of the inventor would be applicable. The invention thus finds use in the presence of proteases, particularly thrombin and Factor Xa.
The assay of the invention finds particular use in the detection of proteases, especially serine proteases and particularly plasmin in milk. Bovine mastitis, inflammation of the mammary gland, is the most common and most costly disease
that afflicts dairy cows world-wide. In mastitis, blood proteins are passively transferred Into the milk as a result of Inflammation. One of these proteins, plasmin, a trypsin-like serine protease, is present at elevated levels in milk while the animal has mastitis. Plasmin has been implicated in the hydrolysis of caseins during Incubation or storage of good quality milk, thereby affecting the quality of dairy products produced. Early detection of plasmin in milk would be of use as an aid in the diagnosis of mastitis, and as an Indicator of the quality of milk for manufacturing. This enables quick and easy rejection of milk which would normally undergo further processing to produce cheese, hence ensuring that only milk of a suitable quality undergoes such further processing.
The peptide inhibitor for the target protease is chosen having regard to the target enzyme that is to be detected. Proteases can be subdivided into general groups depending on their mode of action. Proteases may be described as being trypsin-like, elastase-like or chymotrypsin-like. Trypsin, elastase and chymotrypsin are proteases which have been well-characterised and this description of proteases is well understood in the art. The function of proteases is to fragment a protein by hydrolysing the peptide bonds of that protein.
Trypsin and trypsin-like proteases hydrolyse peptide bonds between either the C-terminal of the ami no acid residues lysine or arginine and the N-terminal of any other amino acid residue. Chymotrypsin and chymotrypsin-like enzymes hydrolyse the peptide bond between the C-terminal of either phenylalanine, tryptophan or tyrosine and the N-terminal of any other amino acid. Elastase and elastase-like enzymes hydrolyse the peptide bond between the C-terminal of valine, alanine or norleucine and the N-terminal of any other amino acid. The protease inhibitor is preferably an irreversible inhibitor.
Proteases may be inhibited by peptides, some of which are known. Preferably the peptide inhibitor of the target enzyme for use in the assay of the invention is a synthetic peptide of 3-20 amino acid residues in length, more preferably of between 3 and
10 amino acid residues and even more preferably 3 and 5 amino acid residues in length. The amino add residues may be naturally occurring amino acids or unnatural amino adds and the Inhibitor is made irreversible by the addition to the C-terminal of the peptide of a chemical group R which is a group capable of modifying the peptide irreversibly, e.g. chloromethyIketone, diphenylphosphorate or fluoromethylketone.
More preferably, the amino acid residue Immediately preceding the R chemical group i.e. the C-terminal amino acid residue of the peptide is a residue "Y" which is the amino acid residue normally recognised by the target protease as the signal for hydrolysis. For trypsin or trypsin-like enzymes, the Y residue
.is preferably an arginine or lysine residue. For chymotrypsin or chymotrypsin-like enzymes, the Y residue is preferably tyrosine, tryptophan or phenylalanine. For elastase or elastase-like enzymes, the Y residue is preferably valine, alanine or norleucine.
In a preferred embodiment of the invention the tracer for the bound target enzyme is preferably a labelled ligand which will usually be a specific binding ligand such as an antibody. Any known method of labelling enzymes for assay purposes may be adopted, e.g. with a detectable marker for example another enzyme, radioisotope, spin label, fluorescent material or the like.
Particularly preferred tracers are polyclonal and monoclonal antibodies raised against the target enzyme or the target enzyme inactivated with the specific inhibitor, with an enzyme label conjugated directly on to the primary antibody or to a secondary antibody directed against the host IgG of an animal in which the primary antibody was raised.
The solid phase can take any suitable form known in heterogeneous assays, including plates, particles, strips, rods, dipsticks, membranes, microtitre wells and so on. Once the target enzyme has been immobilised on the solid phase it can be brought into contact with the tracer, for example a solution of the labelled antibody.
The method of the invention can be used to give a very rapid qualitative analysis of the enzyme under investigation or a quantitative determination. For the latter, mlcrotitre plate reading technology can be applied.
The invention will now be illustrated by way of the following examples:
EXAMPLE 1
Solid phase immunoassav for detection of thermostable serine proteases of bacterial origin in milk
200μl/well of ImM glutaric anhydride was coated onto Nunc Covalink NH microtitre plates overnight at 4°C. The plates were washed twice with distilled water and coated with 100μl/well of ImM 1-ethyl-3 (-3 dimethylaminopropyl) carbodiimide and 100μl/well of 90μM D-Phenylalanyl-L-phenylalanyl-L-arginine chloromethylketone overnight at 4ºC. The plates were washed twice in phosphate buffered saline (PBS) containing 0.05% polyoxyethylenesorbitan monolaurate ("Tween" 20) (PBST) and quenched with PBS containing 0.3% "Tween" 20 for one hour at 37°C. "Tween" is a Registered Trademark.
The plates were washed twice with PBST and 100μl/well of a bacterial protease diluted 1/10 in bovine milk was incubated overnight at 4°C. The plates were washed twice and pre-immune or anti-protease polyclonal antisera were added 100μl/well at a dilution of 1/35 for 1 hour at 37°C. Following five washes, 100μl of goat anti-rabbit Ig conjugated to horse-radish
peroxidase (HRPO) (Sigma) was added at a dilution of 1/1000 for 1 hour at 37°C. The plates were washed five times and o-phenylenediamine (OPD) chromogen containing 0.47. hydrogen peroxide in Sorenson's citrate buffer (0.055M OPD, 0.0285N hydrochloric acid, 0.715M citric acid, 0.143M sodium hydroxide) pH 5.5 was added to each well (100μl) for 30 minutes at room temperature in the dark. The reaction was stopped with 5N H2SO4 (100μl) and the plates were read at 492nm.
The results are set out in Table 1.
TABLE 1
Optical densities obtained i n sol i d phase immunoassay for detection of seri ne proteases in mi lk.
The invention is not restricted to the detection of proteases in milk. It can be used for the detection of enzymes in other complex fluids such as coagulation proteases in blood and tumour specific proteolytic activity. This is outlined in the
following example:
EXAMPLE 2
Solid phase immunoassay for the detection of Plasmin. Factor Xa and Thrombin
COSTAR EIA plates for covalent binding (COOH surface) were coated with 100μl of 1 mM 1-ethyl-3 (-3 dimethylaminopropyl) carbodiimide and 100 μl of peptide inhibitor (see details in Table 2 below) both in distilled water, pH 6.0, overnight at 4°C. Following washing, with acidified water, the remaining reactive COOH sites were 'capped' using 300μl of 27. Glycine methyl ester in distilled water, pH 4.5, for 3 hours at 37ºC. The remaining binding sites on the plates were blocked by the addition of 300μl of a 11 protein solution (bovine serum albumin, ovalbumin or dried milk powder) in distilled water, pH 4.5. Samples containing target enzyme or standards (100μl) were added and incubated for 3 hours at 37°C.
Following washing, with phosphate buffered saline containing 0.057. "Tween" 20, antiserum raised against the target enzyme was added followed by anti-rabbit horseradish peroxidase conjugated antibody. The results were visualised using a chromogenic substrate (OPD or TMB).
The results are shown in Table 2 below. The results for the measurement of bacterial protease found in milk are also included.
Claims
1. A solid phase immunoassay method for detecting a protease in a material, comprising contacting the material under test with a peptide inhibitor for the protease to be detected, said inhibitor being immobilised on a solid phase, whereby the enzyme in the material becomes bound to the solid phase and detecting the bound enzyme with a tracer for the enzyme.
2. An assay according to Claim 1 wherein the material is a biological sample.
3. An assay according to Claim 2 wherein the biological sample is blood.
4. An assay according to any of Claim 3 wherein the protease is thrombin.
5. An assay according to Claim 3 wherein the protease is Factor Xa.
6. An assay according to Claim 2 wherein the biological sample is milk.
7. An assay according to Claim 6 wherein the protease is a serine protease.
8. An assay according to Claim 7 wherein the serine protease is plasmin.
9. An assay according to any of Claims 1 to 8 wherein the peptide inhibitor is irreversible.
10. An assay according to Claim 9 where the peptide inhibitor is made irreversible by the addition to the C-terminal of the peptide of a chemical group R which is a group capable of modifying the peptide irreversibly.
11. An assay according to Claim 10 wherein R is chloromethyl ketone.
12. An assay according to Claim 10 wherein R is diphenylphosphorate.
13. An assay inhibitor according to Claim 10 wherein R is fluoromethylketone.
14. An assay according to any of Claims 9-13 wherein the peptide inhibitor is a synthetic peptide of 3-20 amino acid residues in length wherein the residues may be naturally or unnaturally occurring amino acids.
15. An assay according to Claim 14 wherein the synthetic peptide is 3 to 10 residues in length.
16. An assay according to Claim 15 wherein the synthetic peptide is 3 to 5 residues 1n length.
17. An assay according to any of Claims 9-13 wherein the C-terminal amino add residue "Y" is an amino add residue which is normally recognised by the target protease as the signal for hydrolysis.
18. An assay according to Claim 17 wherein the peptide inhibitor is specific for trypsin or trypsin-like proteases characterised in that Y is an arginine or lysine residue.
19. An assay according to Claim 17 wherein the peptide inhibitor is specific for chymotrypsin or chymotrypsin-like enzymes characterised in that Y is a tyrosine, tryptophan or phenylalanine residue.
20. An assay according to Claim 17 wherein the peptide inhibitor which is specific for elastase or elastase-like enzymes characterised in that the Y residue is a valine, alanine or norleucine residue.
21. An assay according to any preceding Claim wherein the tracer for the bound target enzyme is a labelled ligand.
22. An assay according to Claim 21 in which the labelled ligand is an antibody.
23. An assay according to Claim 22 in which the antibody is polyclonal.
24. An assay according, to Claim 22 in which the antibody is monoclonal.
25. An assay according to any of Claims 21-24 wherein the label is an enzyme.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB919119102A GB9119102D0 (en) | 1991-09-07 | 1991-09-07 | Bioimmunassay |
| GB9119102.3 | 1991-09-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1993005397A1 true WO1993005397A1 (en) | 1993-03-18 |
Family
ID=10701027
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/GB1992/001626 Ceased WO1993005397A1 (en) | 1991-09-07 | 1992-09-07 | Immunoassay for proteases |
Country Status (2)
| Country | Link |
|---|---|
| GB (2) | GB9119102D0 (en) |
| WO (1) | WO1993005397A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0759556A3 (en) * | 1995-07-24 | 1998-05-20 | BEHRINGWERKE Aktiengesellschaft | Method for quantification of activated factors |
| WO2006079826A1 (en) * | 2005-01-28 | 2006-08-03 | Ethicon, Inc. | Device for detecting an enzyme in a sample |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5308755A (en) * | 1992-06-08 | 1994-05-03 | Research Corporation Technologies, Inc. | Method for measuring heparin |
| WO2001038560A2 (en) * | 1999-11-22 | 2001-05-31 | American Red Cross | Active enzyme detection using immobilized enzyme inhibitors |
| GB2435510A (en) | 2006-02-23 | 2007-08-29 | Mologic Ltd | Enzyme detection product and methods |
| GB2435511A (en) | 2006-02-23 | 2007-08-29 | Mologic Ltd | Protease detection |
| GB2435512A (en) | 2006-02-23 | 2007-08-29 | Mologic Ltd | A binding assay and assay device |
| GB2437311A (en) * | 2006-04-07 | 2007-10-24 | Mologic Ltd | A protease detection product |
| WO2012166755A1 (en) * | 2011-06-02 | 2012-12-06 | Lonza Walkersville, Inc. | Enzyme-linked immunosorbent assay for quantitation of residual trypsin in a biological sample |
| GB2504499A (en) * | 2012-07-31 | 2014-02-05 | Baxter Healthcare Sa | Selective measurement of active human protease coagulation factors |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0038935A1 (en) * | 1980-04-30 | 1981-11-04 | MERCK PATENT GmbH | Process for the immunological determination of enzymes, agent for its realization and its use |
| EP0048989A2 (en) * | 1980-09-30 | 1982-04-07 | Cornell Research Foundation, Inc. | Process for determining inhibitor-enzyme complexes |
| EP0080279A1 (en) * | 1981-11-02 | 1983-06-01 | James Walter Ryan | Method for assaying proteases with tagged proteinaceous inhibitors |
| WO1990003577A1 (en) * | 1988-09-30 | 1990-04-05 | The University Of Vermont And State Agricultural College | Immunoassays for catalytically-active, serine proteases |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4753875A (en) * | 1981-11-02 | 1988-06-28 | Ryan James W | Method for assaying proteases with tagged proteinaceous inhibitors |
-
1991
- 1991-09-07 GB GB919119102A patent/GB9119102D0/en active Pending
-
1992
- 1992-09-07 GB GB9218906A patent/GB2259362A/en not_active Withdrawn
- 1992-09-07 WO PCT/GB1992/001626 patent/WO1993005397A1/en not_active Ceased
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0038935A1 (en) * | 1980-04-30 | 1981-11-04 | MERCK PATENT GmbH | Process for the immunological determination of enzymes, agent for its realization and its use |
| EP0048989A2 (en) * | 1980-09-30 | 1982-04-07 | Cornell Research Foundation, Inc. | Process for determining inhibitor-enzyme complexes |
| EP0080279A1 (en) * | 1981-11-02 | 1983-06-01 | James Walter Ryan | Method for assaying proteases with tagged proteinaceous inhibitors |
| WO1990003577A1 (en) * | 1988-09-30 | 1990-04-05 | The University Of Vermont And State Agricultural College | Immunoassays for catalytically-active, serine proteases |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0759556A3 (en) * | 1995-07-24 | 1998-05-20 | BEHRINGWERKE Aktiengesellschaft | Method for quantification of activated factors |
| WO2006079826A1 (en) * | 2005-01-28 | 2006-08-03 | Ethicon, Inc. | Device for detecting an enzyme in a sample |
| GB2435790A (en) * | 2005-01-28 | 2007-09-05 | Ethicon Inc | Device for detecting an enzyme in a sample |
| GB2435790B (en) * | 2005-01-28 | 2009-12-30 | Ethicon Inc | Device for detecting an enzyme in a sample |
Also Published As
| Publication number | Publication date |
|---|---|
| GB9119102D0 (en) | 1991-10-23 |
| GB2259362A (en) | 1993-03-10 |
| GB9218906D0 (en) | 1992-10-21 |
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