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WO1993005397A1 - Dosage immunologique pour proteases - Google Patents

Dosage immunologique pour proteases Download PDF

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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.)
Ceased
Application number
PCT/GB1992/001626
Other languages
English (en)
Inventor
Catherine O'sullivan
Brian Walker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BIOSYN Ltd
Original Assignee
BIOSYN Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BIOSYN Ltd filed Critical BIOSYN Ltd
Publication of WO1993005397A1 publication Critical patent/WO1993005397A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention se rapporte à un procédé de dosage immunologique en phase solide permettant de détecter une protéase dans une matière, consistant à mettre en contact la matière testée avec un inhibiteur peptidique de la protéase à détecter, ledit inhibiteur étant immobilisé sur une phase solide, et l'enzyme de la matière se liant à la phase solide. Ledit procédé consiste également à détecter l'enzyme liée, avec un traceur d'enzyme.
PCT/GB1992/001626 1991-09-07 1992-09-07 Dosage immunologique pour proteases Ceased WO1993005397A1 (fr)

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 (fr) 1993-03-18

Family

ID=10701027

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1992/001626 Ceased WO1993005397A1 (fr) 1991-09-07 1992-09-07 Dosage immunologique pour proteases

Country Status (2)

Country Link
GB (2) GB9119102D0 (fr)
WO (1) WO1993005397A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0759556A3 (fr) * 1995-07-24 1998-05-20 BEHRINGWERKE Aktiengesellschaft Méthode pour la quantification des facteurs actifs
WO2006079826A1 (fr) * 2005-01-28 2006-08-03 Ethicon, Inc. Dispositif de détection d’une enzyme dans un échantillon

Families Citing this family (8)

* Cited by examiner, † Cited by third party
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 (fr) * 1999-11-22 2001-05-31 American Red Cross Nouvelle methode permettant de detecter la presence d'une forme d'enzyme fonctionnellement active dans des echantillons biologiques et kit
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 (fr) * 2011-06-02 2012-12-06 Lonza Walkersville, Inc. Essai par immunosorbant lié à une enzyme pour la quantification de trypsine résiduelle dans un échantillon biologique
GB2504499A (en) * 2012-07-31 2014-02-05 Baxter Healthcare Sa Selective measurement of active human protease coagulation factors

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0038935A1 (fr) * 1980-04-30 1981-11-04 MERCK PATENT GmbH Procédé pour la détermination immunologique d'enzymes, agents pour sa mise en oeuvre et son utilisation
EP0048989A2 (fr) * 1980-09-30 1982-04-07 Cornell Research Foundation, Inc. Procédé pour la détermination des complexes inhibiteurs d'enzymes
EP0080279A1 (fr) * 1981-11-02 1983-06-01 James Walter Ryan Procédé pour l'essayage de protéinase avec inhibiteurs protéidiques marqués
WO1990003577A1 (fr) * 1988-09-30 1990-04-05 The University Of Vermont And State Agricultural College Immunoanalyses pour la detection de proteases de serine presentant une activite catalytique

Family Cites Families (1)

* Cited by examiner, † Cited by third party
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

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0038935A1 (fr) * 1980-04-30 1981-11-04 MERCK PATENT GmbH Procédé pour la détermination immunologique d'enzymes, agents pour sa mise en oeuvre et son utilisation
EP0048989A2 (fr) * 1980-09-30 1982-04-07 Cornell Research Foundation, Inc. Procédé pour la détermination des complexes inhibiteurs d'enzymes
EP0080279A1 (fr) * 1981-11-02 1983-06-01 James Walter Ryan Procédé pour l'essayage de protéinase avec inhibiteurs protéidiques marqués
WO1990003577A1 (fr) * 1988-09-30 1990-04-05 The University Of Vermont And State Agricultural College Immunoanalyses pour la detection de proteases de serine presentant une activite catalytique

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0759556A3 (fr) * 1995-07-24 1998-05-20 BEHRINGWERKE Aktiengesellschaft Méthode pour la quantification des facteurs actifs
WO2006079826A1 (fr) * 2005-01-28 2006-08-03 Ethicon, Inc. Dispositif de détection d’une enzyme dans un échantillon
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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