GB2259362A - Bio immunoassay for proteases - Google Patents

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. The biological sample may be blood or milk. The protease to be detected may be thrombin, Factor Xa, a serine protease or plasmin. The inhibitor may be specific for trypsin, chymotrypsin or elastase enzymes. The inhibitor may comprise chloromethylketone, diphenylphosphate or fluoromethylketone. The solid phase can take any suitable form e.g. plates, particles, strips, rods, dipsticks, membranes, microtitre wells. The tracer may be a monoclonal or polyclonal antibody labelled with, for example, another enzyme, radioisotope, spin label or fluorescent materials.

Description

BIO IMMUNOASSAY 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 cr 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. Plasmln has been mpl 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 amino 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 acid residues may be naturally occurring amino acids or unnatural amino acids 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. chloromethylketone, di phenyl phosphorate or fl uoromethyl ketone.

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 lgG 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, microtitre plate reading technology can be applied.

The invention will now be illustrated by way of the following examples: EXAMPLE 1 Solid phase immunoassay for detection of thermostable serine Droteases of bacterial origin in milk 200p1/well of lmM 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 100p1/well of lmM l-ethyl-3 (-3 dimethylaminopropyl) carbodiimide and 1001/ell of 90jiM D-Phenylalanyl-L-phenylalanyl-L-arginine chloromethylketone overnight at 4"C. The plates were washed twice in phosphate buffered saline (PBS) containing 0.05% polyoxyethylenesorbitan monolaurate ("Tween" 20) (PBST) and quenched with PBS containing 0.3X "Tween" 20 for one hour at 37cC. "Tween" is a Registered Trademark.

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 100p1/well at a dilution of 1/35 for 1 hour at 37"C. Following five washes, 100p1 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 0.4% 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 (100p1) for 30 minutes at room temperature in the dark. The reaction was stopped with SN H2S04 (100p1) and the plates were read at 492nm.

The results are set out in Table 1.

TABLE 1 Optical densities obtained in solid phase immunoassay for detection of serine proteases in milk.

Optical Density 492nm CONTROL Pre-immune antiserum 0.228 + 0.04 TEST Anti-proteinase antiserum 1.302 + 0.06 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 100p1 of 1 mM l-ethyl-3 (-3 dimethylaminopropyl) carbodiimide and 100 pl of peptide inhibitor (see details in Table 2 below) both in distilled water, pH 6.0, overnight at 40C.Following washing, with acidified water, the remaining reactive C00H sites were 'capped' using 300p1 of 2% Glycine methyl ester in distilled water, pH 4.5, for 3 hours at 37"C.

The remaining binding sites on the plates were blocked by the addition of 300p1 of a 1% protein solution (bovine serum albumin, ovalbumin or dried milk powder) in distilled water, pH 4.5.

Samples containing target enzyme or standards (100p1) were added and incubated for 3 hours at 37"C.

Following washing, with phosphate buffered saline containing 0.05% "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.

TABLE 2 ENZYME INHIBITOR Concentration Current assay used in assay sensitivity Thrombin D-Phe-Pro-Arg- 1 pM 25 pM in PBS CMK Factor Xa Ile-Glu-Gly- 1-100 pM 1 pg/ml in PBS Arg-CMK assay development just beginning.

Plasmin D-Val-Phe-Lys- 1-100pM < 1 ng/ml in milk CMK Bacterial D-Phe-Phe-Arg- 1-100pM Difficult to Protease CMK determine - no known standard.

Claims (25)

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 thrombi n.

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 chl oromethyl ketone.

12. An assay according to Claim 10 wherein R is di phenyl phosphorate.

13. An assay inhibitor according to Claim 10 wherein R is fl uoromethyl ketone.

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 In length.

17. An assay according to any of Claims 9-13 wherein the C-terminal amino acid residue "Y" is an amino acid 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.