# Patent

## Title
Diagnostic test

## Abstract

An assay for detecting type IV cytosolic phospholipase A2(cPLA2) or a protein immunologically homologous to type IV cPLA2, the assay comprising use of red blood cells, particularly for use in the diagnosis of a disease in which dysfunction of cell signalling systems involving highly unsaturated fatty acids is implicated.

## Background
The present invention is concerned with identification of proteins of the cytosolic phospholipase A2 enzyme and the applications thereof, particularly in diagnosis, treatment monitoring and drug development.
Phospholipase A2 (PLA2) enzymes are generally characterised by their capacity to catalyse the hydrolysis of the Sn2 acyl-ester bond of glycerophosphate to release free fatty acid (Mayer R. J. and Marshall L., FASEB J, 7, 339–348; Dennis E. A., Ed., Phospholipase A2 Methods in Enzymology, 1991, 197, 359–433). The scientific literature recognises different types of PLA2 enzyme (Dennis E. A., Trends Biochem. Sci., 22, 1–2). Types I, II and III are referred to as secretory phospholipase A2 (sPLA2) enzymes. Secretory phospholipase A2 enzymes have a molecular weight of approximately 14 kDa, are found extracellularly and have been recognised in plasma.
The type IV PLA2 enzyme is referred to as cytosolic phospholipase A2 (cPLA2). It features a calcium binding domain, termed the C2 domain, and its phospholipase activity is highly selective for highly-unsaturated fatty acids, particularly arachidonic acid, present in the Sn2 acyl-ester glycerophosphate bond. Its ability to release arachidonic acid from cell membrane phospholipids sets it in a key position for the regulation of the supply of arachidonate and subsequent action in cell messenger processes.
A further type of PLA2 enzyme is the type VI enzyme which is also a cytosolic PLA2 enzyme, but which does not possess the high selectivity for arachidonic acid in the Sn2 position of glycerophospate for phospholipid acyl ester hydrolysis (Tang J. et al., J. Biol. Chem., 272, 1997, 8567–8575). It is termed iPLA2 in recognition that it is independent of calcium level for its catalytic activity.
The type IV arachidonate-selective cPLA2 enzyme has been identified in a number of tissues including human monocytes (Clark J. D. et al., Cell, 65, 1991, 1043–1051; Kramer R. M. et al., J. Biol. Chem., 266, 1991, 5268–5272) and human platelets (Takayama K. et al., FEBS, 282, 1991, 326–330).
The amino acid sequence of type IV cPLA2 has been determined for the enzyme purified from human monocyte (U937) cells (Clark J. D. et al., Cell, 65, 1991, 1043–1051). Type IV cPLA2 found in U937 cells has an amino acid sequence which is 749 amino acids long. This sequence shows a region of homology with a limited number of other proteins including protein kinase C (PKC), GTPase activating protein (GAP), phospholipase C and synaptic vesicle protein p65. The points of homology occur towards the N-terminal end of the protein in the so-called calcium-binding portion which is at or around amino acids 36 to 81 of the sequence (counting from the NH2 terminal end) (Nalefski et al., J. Biol. Chem., 269, 18239–18249). There are no known areas of sequence homology between other mammalian proteins and type IV cPLA2 amino acids 82 to 749, apart from a sequence from amino acids 129 to 135 shared with pulmonary surfactant protein C (Clark et al., J. Lipid Mediators Cell Signalling, 12, 83–117) and two recently reported cytosolic phospholipase A2 enzymes designated cPLA2β and cPLA2γ (R. T. Pickard et al, J. Biol. Chem., 1999, 274, 8823–8831). These are proteins different from the cPLA2 enzyme isolated from human U937 monocytes (which now may be designated as cPLA2α). The cPLA2α, β and γ sequence homology is limited to five amino acids found in cPLA2α at position serine 228 to tryptophan 232.
The catalytic active centre of type IV cPLA2 has been reported as being located in a peptide sequence comprising amino acid 228 (Clark J. D. et al., J. Lipid Mediators Cell Signalling, 12, 83–117) and has recently been described as being dependent for its function on the proximity of two amino acids brought together in the folded tertiary structure of the molecule, serine 228 and aspartate 549 (Dessen A. et al., Cell, 1999, 97, 349–360).
Previous observations of PLA2 enzymes in the circulation have been directed at measurements in serum or plasma by substrate assay methods and have not characterised the type of the enzyme involved (Thuren T. et al., Clin. Chem., 31, 1985, 714–717; Gattaz W. F. et al., Biol. Psychiatry, 22, 1987, 421–426; Gattaz W. F. et al., Biol. Psychiatry, 28, 1990, 495–501). Serum and plasma PLA2 enzymes show increased activity relative to normal control subjects in the human disease schizophrenia, although again the PLA2 type of the enzyme responsible has not been characterised. To date, there has been no identification of cytosolic phospholipase A2 type IV protein either in or attached to red cells either physiologically or in pathology.
It has now been found that type IV cPLA2 proteins or proteins immunologically homologous to type IV cPLA2 can be detected in or on circulating red blood cells. The detection and assay of these cPLA2 proteins has application in the diagnosis of disease, in the monitoring of the patient response to treatment and in the development of drugs which influence the activity or concentration of cPLA2. Recognition that these cPLA2 proteins may be detected in or on red blood cells provides a simple and convenient method of assaying for the cPLA2 level.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts western blots. FIG. 1a depicts a western blot to identify type IV cPLA2 antigen using a rabbit polyclonal antibody raised against mid-molecule type IV cPLA2 and a swine anti-rabbit IgG antibody coupled to horseradish peroxidase. Lane 1: molecular weight markers. Lane 2: cell cytosol from insect cells infected with baculovirus expressing type IV cPLA2. Lanes 3–12: samples of red cell hemolysates from schizophrenic patients taking clozapine (lanes 4, 7, 9 and 10), schizophrenic patients taking neuroleptic drugs (lanes 8 and 12), and control patients (lanes 3, 5, 6 and 11). FIG. 1b depicts a western blot to identify antigens other than type IV cPLA2 and a swine anti-rabbit IgG antibody coupled to horseradish peroxidase. Lane 1: molecular weight markers. Lane 2: cell cytosol from insect cells infected with baculovirus expressing type IV cPLA2. Lanes 3–12: samples of red cell hemolysates taken schizophrenic patients taking clozapine (lanes 4, 7, 9 and 10), schizophrenic patients taking neuroleptic drugs (lanes 8 and 12) and control patients (lanes 3, 5, 6 and 11).
FIG. 2 depicts a calibration curve for an enzyme-linked immunoassay for type IV cPLA2 protein, which uses a luminescence end point for protein detection.
FIG. 3 depicts a frequency histogram of type IV cPLA2 levels. FIG. 3A depicts a frequency histogram of type IV cPLA2 levels in the red blood cells of 51 healthy volunteers using an enzyme-linked immunoassay for type IV cPLA2 protein. FIG. 3B depicts a frequency histogram of type IV cPLA2 levels in the red blood cells of 49 patients who meet the DSM IV criteria for the diagnosis of schizophrenia using an enzyme-linked immunoassay for type IV cPLA2 protein. FIG. 3C depicts a frequency histogram of type IV cPLA2 levels in the red blood cells of 27 volunteers with recognized criteria for identification of dyslexia using an enzyme-linked immunoassay for type IV cPLA2 protein.
FIG. 4 demonstrates a western blot analysis of type IV cPLA2 protein antigen in red cell hemolysates from a schizophrenia subject and a control subject. Lane 1: molecular weight markers. Lane 2: cytosol from insect cells infected with baculovirus expressing type IV cPLA2. Lane 3: cytosol form human monocyte U937 cells containing a cPLA2 protein. Lane 4: red cell hemolysate from a control subject. Lane 5: red cell hemolysate from a schizophrenia subject.



According to a first aspect of the present invention, there is provided an assay for detecting type IV cPLA2 or a protein immunologically homologous to type IV cPLA2, the assay comprising use of red blood cells. In a second aspect of the invention, there is provided an assay for quantifying the level of type IV cPLA2 or a protein immunologically homologous to type IV cPLA2 in a sample of red blood cells. The assay may be performed on a sample of whole blood or on a sample of red blood cells after separation thereof from whole blood.
As used herein, the term “a protein immunologically homologous to type IV cPLA2” means a protein that binds specifically to an antibody or antibodies that recognise an epitope or epitopes from any part of type IV cPLA2 protein from human monocyte (U937) cells, and preferably an epitope or epitopes from amino acids 82 to 749 of type IV cPLA2 protein from human monocyte (U937) cells. In a preferred embodiment of the invention, the term “a protein immunologically homologous to type IV cPLA2” means a protein that binds specifically to an antibody or antibodies that recognise an epitope or epitopes from a peptide sequence or sequences which comprise the catalytic active centre of type IV cPLA2 protein from human monocyte (U937) cells. In a further preferred embodiment, the term “a protein immunologically homologous to type IV cPLA2” means a protein that binds specifically to an antibody or antibodies that recognise an epitope or epitopes from the peptide sequence of amino acids 241 to 260 of type IV cPLA2 protein from human monocyte (U937) cells.
In an alternative embodiment, the term “a protein immunologically homologous to type IV cPLA2” means a protein that binds specifically to an antibody or antibodies raised against an epitope or epitopes from any part of type IV cPLA2 protein from human monocyte (U937) cells or a synthetic peptide matching the sequence of type IV cPLA2 protein from human monocyte (U937) cells. In a preferred alternative embodiment, the term “a protein immunologically homologous to type IV cPLA2” means a protein that binds specifically to an antibody or antibodies raised against an epitope or epitopes from amino acids 82 to 749, and preferably an epitope or epitopes from a peptide sequence or sequences which comprises the catalytic active centre, of type IV cPLA2 protein from human monocyte (U937) cells or a synthetic peptide matching the sequence of amino acids 82 to 749, and preferably a sequence or sequences which comprises the catalytic active centre, of type IV cPLA2 protein from human monocyte (U937) cells. In a further alternative embodiment, the term “a protein immunologically homologous to type IV cPLA2” means a protein that binds specifically to an antibody or antibodies raised against an epitope or epitopes from the peptide sequence of amino acids 241 to 260 of type IV cPLA2 protein from human monocyte (U937) cells, or a synthetic peptide matching the sequence of amino acids from the peptide sequence of amino acids 241 to 260 of type IV cPLA2 protein from human monocyte (U937) cells.
According to a third aspect of the present invention, there is provided a method of diagnosis of a disease in which dysfunction of cell signalling systems involving highly unsaturated fatty acids is implicated, said method comprising the detection of type IV cPLA2 protein or a protein immunologically homologous to type IV cPLA2 in or on red blood cells. The method may further comprise the step of determining the level of type IV cPLA2 protein or a protein immunologically homologous to type TV cPLA2 in or on red blood cells and, optimally, comparing this level with a control level.
As used herein, the term “highly unsaturated fatty acids” includes all fatty acids released by action of the type IV cPLA2 enzyme. In an embodiment of the invention the term “highly unsaturated fatty acids” includes fatty acids having 3 or more carbon—carbon double bonds. In particular, the term includes the essential fatty acids, particularly the fatty acids of the group comprising dihomogammalinolenic acid (DGLA; 8,11,14-eicosatrienoic acid), arachidonic acid (AA; 5,8,11,14-eicosatetraenoic acid), adrenic acid (7,10,13,16-docosatetraenoic acid), 4,7,10,13,16-docosapentaenoic acid, stearidonic acid (SA; 6,9,12,15-octadecatetraenoic acid), 8,11,14,17-eicosatetraenoic acid, eicosapentaenoic acid (EPA; 5,8,11,14,17-eicosapentaenoic acid), docosapentaenoic acid (DPA; 7,10,13,16,19docosapentaenoic acid) and docosahexaenoic acid (DHA; 4,7,10,13,16,19-docosahexaenoic acid).
According to a fourth aspect of the invention, there is provided a method of monitoring the effectiveness of medication administered to a patient suffering from a disease in which dysfunction of cell signalling systems involving highly unsaturated fatty acids is implicated, said method comprising the detection of type IV cPLA2 protein or a protein immunologically homologous to type TV cPLA2 in or on red blood cells. The method may, for example, comprise the steps of administering to a patient a compound to be tested, determining the level of type IV cPLA2 protein or a protein immunologically homologous to type IV cPLA2 in or on red blood cells and, optimally, comparing this level with a control level or with a level or levels determined at an earlier stage of the medication regime or before the medication regime commenced.
According to a fifth aspect of the invention, there is provided a method of drug development for a disease in which dysfunction of the cell signalling systems involving highly unsaturated fatty acids is implicated, said method comprising the detection of type IV cPLA2 protein or a protein immunologically homologous to type IV cPLA2 in or on red blood cells. The method may, for example, be used in the screening of compounds for use in the treatment of a disease in which dysfunction of cell signalling systems involving highly unsaturated fatty acids is implicated, the method comprising the steps of administering to a patient or test animal a compound to be tested, determining the level of type IV cPLA2 protein or a protein immunologically homologous to type IV cPLA2 in or on red blood cells and, optimally, comparing the level with a control level.
The invention is particularly concerned with diseases in which dysfunction of cell signalling systems involving arachidonic acid, dihomogammalinolenic acid, eicosapentaenoic acid, docosapentaenoic acid and/or docosahexaenoic acid is implicated. More particularly, the invention is concerned with diseases in which dysfunction of cell signalling systems involving arachidonic acid is implicated.
The disease conditions in which the identification of type IV cPLA2 proteins or proteins immunologically homologous to type IV cPLA2 in or on red cells may be useful include:

    1) schizophrenia, in which increased cPLA2 expression and activity is a proposed mechanism in disease development;
    2) bipolar or manic depressive illness, in which cPLA2 abnormality may be present;
    3) cachexia, in which tumour necrosis factor promotes cPLA2 activity;
    4) brain injury, including stroke and mechanical injury, in which cPLA2 may be released from damaged membranes or as part of the process of apoptosis;
    5) dyslexia, in which abnormal cPLA2 activity may be present; and
    6) any other disease or disease process in which type IV cPLA2 activity or concentration is increased or decreased from normal levels, particularly a disease or disease process in which type IV cPLA2 activity or concentration is increased.


The assays and methods of the invention may comprise the steps of collecting a sample of blood from a subject, and detecting the proteins ex vivo. Preferably, the assays and methods further comprise one or more of the steps of separating the red cells from the other blood components, disrupting the red cells by a method such as sonication, nitrogen cavitation, freezing or lysis, and detecting the proteins either directly or following a protein separation technique. Another application for the assays and methods of the invention include their employment on whole blood, with or without the requirement of prior separation of the red cells, and such applications would include, for example, a near-patient testing diagnostic strip, cartridge, or device.
In a preferred embodiment, the assays and methods of the invention comprise the detection in or on red blood cells of one or more proteins from one or more of three particular groups of proteins. The proteins are grouped by apparent molecular weight as measured using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) with western blot and can be designated as follows:

    Group A, comprising one or more proteins of apparent molecular weight in the range of 80 to 110 kDa, particularly in the range of 90 to 105 kDa;
    Group B, comprising more or more proteins of apparent molecular weight in the range of 70 to 80 kDa;
    Group C, comprising one or more proteins of apparent molecular weight in the range of 50 to 60 kDa.


It is considered that the proteins in a given group may be structurally related, small variations in apparent molecular weight between the proteins in a given group possibly being due to the extent of phosphorylation. However, it is not intended that the scope of the invention be limited by this theory. Variations in apparent molecular weight among proteins of a given group may simply be due to variations in the length of the amino acid sequence of the protein or protein fragment.
These proteins react immunologically with antibodies to cPLA2. The proteins can be identified by an antibody or antibodies which react with an epitope or epitopes present in a region of the type IV cPLA2 protein which does not share sequence homology with other proteins, for example a region other than the calcium-binding portion of the protein. The proteins can also be identified by using a first antibody (a “capture antibody”) which reacts with an epitope or epitopes present in a sequence having homology with other proteins, for example, the calcium binding portion of the type IV cPLA2 protein, and a second antibody (a “detection antibody”) which reacts with an epitope or epitopes in a region of the protein which does not have homology with other proteins in order to provide the necessary specificity. Protein detection and identification is discussed in more detail below.
It is considered that the proteins or protein fragments isolated from in or on red blood cells may be the same as the type TV cPLA2 protein, or fragments thereof, found in human monocyte (U937) cells. Thus, it is considered that the proteins having apparent molecular weights in the range of 80 to 110 kDa (particularly 90 to 105 kDa), in the range 70 to 80 kDa and in the range 50 to 60 kDa may be either intact type TV cPLA2 or major components of type IV cPLA2. However, it is not intended that the scope of the invention be limited by this theory.
According to a further aspect of the invention there is provided a protein obtainable by isolation from red blood cells, said protein being immunologically homologous to type IV cPLA2. Said protein may exist in the body in the form of a protein having a molecular weight in the range 80 to 110 kDa (and particularly in the range of 90 to 105 kDa) or in the form of a protein having a molecular weight in the range 70 to 80 kDa or in the form of a protein having a molecular weight in the range 50 to 60 kDa.
Protein Separation
The type IV cPLA2 proteins or proteins immunologically homologous to type IV cPLA2 proteins are typically separated from the other proteins of red blood cells by SDS PAGE but a variety of other protein separation techniques may be used, including native polyacrylamide gel electrophoresis and various column separation techniques such as fast protein liquid chromatography and variants of Sepharose or other commercially available columns. Two dimensional electrophoretic techniques may also be used. As noted above, prior protein separation is not always necessary and the type IV cPLA2 proteins or proteins immunologically homologous to type IV cPLA2 may be detected in red cells by immunological means without the necessity of prior protein separation.
Protein Detection
The detection of type IV cPLA2 proteins and/or proteins immunologically homologous to type IV cPLA2 can be achieved by a wide range of protein detection procedures including:

    1) enzyme linked immunoassay, radioimmunoassay, luminescence immunoassay, fluorescence immunoassay or any other variant of competitive or tagged antibody immunoassay, including biosensor technology (evanescent wave optical biosensor);
    2) immunofluoroescence or other immunoassay on red blood cells in smears, droplets, films, fixed histological sections or tissue samples;
    3) in vitro tests in which the protein is recognised by a specific polyclonal or monoclonal antibody and the specific antibody is itself recognised in a Coombs-type test; and
    4) enzymic substrate assay of type IV cPLA2 proteins utilising natural or artificial phospholipid substrates together with specific inhibitors of the other phospholipase enzymes.


In one embodiment of the invention, protein detection is achieved using diagnostic strips, cartridges or devices suitable for near-patient testing which detect the proteins using any of the above techniques or any other technique known in the art.
In a preferred embodiment, the proteins are detected using either a polyclonal or a monoclonal antibody or a combination thereof. Table 1 provides examples of antibodies suitable for use in the assays of the present invention for the detection of type IV cPLA2 proteins or proteins immunologically homologous to type IV cPLA2.
Suitable antibodies or combinations of antibodies for use in the present invention can also include one or more of, for example, a polyclonal antibody raised against an epitope of the peptide chain comprising amino acids 730 to 749 of type IV cPLA2 from U937 cells, or an extended or different amino acid sequence from the C-terminal end of the molecule; an antibody or antibodies raised against a mid-molecule peptide sequence or sequences which comprise the catalytic active site of type IV cPLA2 from U937 cells; an antibody or antibodies raised against an epitope from the N-terminal part of the sequence (amino acids 1 to 216); and an antibody or antibodies raised against another epitope or epitopes from the amino acid sequence of type IV cPLA2 protein from U937 cells between amino acids 82 and 749.











TABLE 1









DETECTS cPLA2-




PRODUCTION

LIKE PROTEINS


ANTIGEN
TYPE
METHOD
SOURCE
IN RED CELLS







Human type IV cPLA2
Polyclonal IgG
Raised in Rabbits
Cayman
Yes


synthetic peptide from


Chemical


mid molecule


Human type IV cPLA2
Polyclonal IgG
Raised in Sheep
The Binding
Yes


synthetic peptide of a 24


Site


amino acid sequence from


the C-terminal domain


Human cPLA2 synthetic
Monoclonal IgG
Mouse
Chemicon
Yes


peptide to amino terminal


International


(amino acids 1–216)


Inc.


Human type IV cPLA2
Polyclonal IgG
Raised in Sheep
In-house
Yes


synthetic peptide mid


production


molecule sequence (amino


acids 241 to 260)a





a: This antibody is prepared using as antigen a synthetic peptide comprising amino acids 241 to 260 of type IV cPLA2 (as found in human monocytes, U937 cells), coupled to ovalbumin. The antigen is injected into sheep with Freunds adjuvant combining the intra-muscular and subcutaneous routes in standard approved protocol. The resulting antiserum was purified initially using Protein A and further purified using an affinity matrix carrying the peptide 241 to 260 not coupled to ovalbumin, to provide an affinity purified IgG antibody.






The recognition of type IV cPLA2 proteins or proteins immunologically homologous to type IV cPLA2 in an accessible component of blood opens the way to their use as follows:

    a) Diagnostic use by direct immunoassay of their level.
    b) Use in a diagnostic sense indirectly as part of a Coombs-type test.
    c) Recognition by immunofluorescence on or in red blood cells on slide or smear or tissue preparation.
    d) Use in an immune precipitation or other reaction to recognise type IV cPLA2 proteins or proteins immunologically homologous to type IV cPLA2 in whole blood samples in near-patient testing.
    e) Use in monitoring of treatments which are directed to suppress PLA2 activity or concentration.
    f) Use in the research for agents which suppress PLA2 activity or concentration for drug discovery.


The invention will now be described in detail with reference to the following examples. It will be appreciated that the invention is described by way of example and modification of detail may be made without departing from the scope of the invention.
EXAMPLES
I. Collection of Samples
The collection of samples for protein separation and detection were performed as follows:

    1) Samples of venous blood (4 to 6 ml) were collected by venepuncture using EDTA at standard concentration for anticoagulation (Vacutainer® or Starstedt Monovette ®). Other appropriate anticoagulants known to persons skilled in the art may also be used.)
    2) Within 3 minutes of the sample being withdrawn, protease inhibitors were added. To the 4 or 6 ml volume of blood were added 0.5 ml of a protease inhibitor cocktail, freshly prepared, as follows:
    
        To 3 mls of Aprotinin (Trasylol®) concentration 10.000 Kallikrein inactivator units per ml was added 2 mg phenylmethylsulphonyl fluoride (PMSF) and 0.5 mg leupeptin. Other appropriate inhibitor preparations known to persons skilled in the art may also be used.
    
    
    3) The whole blood sample was then centrifuged, at 3000 rpm (1,000 g), for 10 minutes. Plasma was removed and the layer of white cells and platelets (the buffy coat) removed by plastic pipette, to leave the red cells in the tube.
    4) A volume of ice cold phosphate buffered saline PBS pH 7.4 or other appropriate buffer, equal in volume to the red cells was added and red cells and buffer were mixed by inversion.
    5) The cells in buffer were then centrifuged, at 1,000 g, repeating steps 3 and 4 three times, providing washed red cells.
    6) The washed red cells were frozen in 0.5 ml aliquots were suspended in 0.5 ml of buffer, prepared as follows: 0.37 g KCl, 0.74 g disodium EDTA, 3.0 g Tris, 9 g NaCl in 1 liter of water, adjusted to pH 7.4 with HCl. (Kramer et al. J. Biol. Chem. 266, 5268–5272). The washed red cells may alternatively be frozen directly as packed cells.
    
        In a simplified procedure, blood is collected into an EDTA vacutainer, centrifuged at 3000 rpm (1000 g) for 10 minutes, the plasma and the buffy coat removed by aspiration and the packed red cells stored frozen.
    
    
    7) These samples were stored frozen at −80° C. or used directly after brief freezing to provide lysis for SDS polyacrylamide gel electrophoresis or for immunoassay in a calibrated assay. Where packed cells are stored frozen prior to use they are thawed into the buffer described in step (6) above.
    
        Where the simplified procedure is employed, protease inhibitor is included in the buffer added to the red cells for the thawing process in the form of one tablet of COMPLETETM protease inhibitor (Boehringer Mannhein) per 200 ml of buffer.

I. Protein Separation and Detection using SDS PAGE

    
    


Protein separation and detection was achieved using SDS PAGE as described below.
In SDS PAGE, the sample was treated with a volume of sample buffer, comprising Tris buffer pH 6.8 containing 6% SDS, 0.5% dithiothreitol and 20 ml glycerol per 100 ml. (Alternative buffers known to persons skilled in the art may be used). Lysate was added to give a 4-fold or greater dilution of the lysate in sample buffer. The sample was transferred to the standard SDS 7.5% polyacrylamide gel for electrophoresis. After electrophoresis the proteins were transferred from the gel to a membrane by electroblotting.
The test membrane was reacted with polyclonal antibody to mid-molecule type IV cPLA2 raised in rabbit and then anti rabbit IgG antibody raised in swine which has been coupled to horse raddish peroxidase to demonstrate the presence of the cPLA2 antigen. The blank membrane was reacted with anti rabbit IgG antibody raised in swine which has been coupled to horse raddish peroxidase. Any bands observed on the blank membrane are due to the presence of antigens other than the cPLA2 antigen.
FIG. 1a and lb show two western blot analyses (FIG. 1a is the test membrane; FIG. 1b is the blank membrane) on the following samples:

    Lane 1: Molecular weight markers
    Lane 2: Cell cytosol from insect cells infected with Baculovirus expressing type TV cPLA2.
    Lanes 3–12: Patient samples. The samples were taken from schizophrenic patients on clozapine (lanes 4, 7, 9 and 10), schizophrenic patients on neuroleptic drugs (lanes 8 and 12) and from control patients (lanes 3, 5, 6 and 11).


The patient samples on the test membrane show three band types grouped by molecular weight as described above:

    Group A: one band is observed above the 97 kDa molecular weight marker, having a molecular weight of about 100 to 105 kDa, and one band is observed below the 97 kDa molecular weight marker, having a molecular weight of about 90 to 95 kDa;
    Group B: a single band is observed with molecular weight around 70 kDa;
    Group C: three bands are observed with molecular weights around 60 kDa. Two of the bands in this molecular weight group are believed to be type IV cPLA2 proteins. The most distinct of these also appears on the blank membrane, has a less specific immunological response and may not be a type IV cPLA2 protein.


In summary, the red cell lysates contained five proteins, two in the range of about 90 to about 105 kDa, one in the range of about 70 to about 80 kDa, and two in the range of about 50 to about 60 kDa, which reacted specifically in SDS PAGE western blot analysis with polyclonal antibody prepared against mid-molecule sequences of type IV cPLA2.
As noted above the bands grouped in Groups A, B and C may represent, respectively, the intact type IV cPLA2 molecule in differing stages of phosphorylation and two groups of peptide sequences of type IV cPLA2 proteins which have lost amino acids by protein degradation. However, as stated above, it is not intended that the scope of the invention be limited by this theory. The bands grouped in Groups A, B and C could also represent intact type IV cPLA2 and fragments of type IV cPLA2 differing in the length of amino acid sequence.
III. Protein Detection Using Enzyme Linked Immunoassay (ELISA)
Immunoassay, specifically enzyme linked immunoassay (ELISA), was also used to detect red cell type IV cPLA2 proteins or proteins immunologically homologous to type IV cPLA2. This method uses a luminescence end point for protein detection.
Samples are collected and prepared as hereinbefore described. Samples of red cells for analysis were stored at −80° C.
The following reagents are required for ELISA:

    1) Coating buffer, pH 9.6
    
        1.59 g sodium carbonate, 2.93 g sodium bicarbonate in 1 liter deionised water;
    
    
    2) Wash buffer, pH 7.4
    
        6.07 g Tris, 0.2 g potassium chloride, 29.2 g sodium chloride in 800 ml deionised water adjusted to pH 7.4 with hydrochloric acid and made up to 1 liter;
    
    
    3) Buffer for standard/sample diluent, pH 7.4
    
        0.37 g potassium chloride, 0.74 g disodium ethylenediaminetetra-acetate, 3 g Tris, 8.58 g sodium chloride in 800 mls deionised water adjusted to pH 7.4 and made up to 1 liter;
    
    
    4) Standard/sample diluent
    
        200 ml of buffer (3) to which is added 0.2 g human albumin and 1 tablet of Complete® protease inhibitors (Boehringer Mannheim);
    
    
    5) Citrate solution, pH 7.4
    
        2.94 sodium citrate dissolved in 800 ml deionised water adjusted to pH 7.4 with hydrochloric acid and made up to 1 liter;
    
    
    6) Antibody conjugate reagent
    
        5 mls reagent (2), 6 mls Roti-Block® (Rotech Scientific, Milton Keynes, UK), 0.2 ml reagent (5), 0.012 g human serum albumin and 1 ml of the in-house anti-cPLA2 IgG (as described herein)—alkaline phosphatase conjugate;
    
    
    7) Luminescence reagent containing Lumi-Phos® 530 (buffered solution containing dioxetane Lumigen® PPD, fluorescer and surfactant) (obtained from Beckman Coulter, UK)


The assay is performed using 96 well microtitre plates according to the following procedure:

    1) The capture antibody (obtained from The Binding Site. Birmingham. UK) was diluted 1:10 with saline and stored in 500 μl aliquots at −20° C. One aliquot of this antibody is added to 11.5 ml coating buffer, mixed and 100 μl of diluted capture antibody is pipetted into each well of a white microtitre plate to coat the wells of the plate with capture antibody. The plate is sealed and stored at 4° C. overnight. Prior to use, the plate is then washed four times with wash buffer pH 7.4 (reagent (2) as hereinbefore described).
    2) The plate is blocked by pipetting into each well 300 μl of Roti-Block® diluted 1:2 with reagent (2). Blocking takes 90 minutes at 25° C. Blocking reagent is removed by washing four times with reagent (2).
    3) Duplicate aliquots (100 μl) of sample or standard are then pipetted into each microtitre plate well and the plate incubated for 90 minutes at 25° C. The incubation is stopped by washing the plate four times with reagent (2).
    4) The second antibody (the conjugated antibody, reagent (6)) is then added. Antibody is conjugated to alkaline phosphatase by standard techniques (Duncan et al., Analytical Biochemistry 132, 68–73). 100 μl of the antibody conjugate, reagent (6), is added to each well and the plate incubated for 90 minutes at 25° C.
    5) At the end of this incubation, the plate is washed 8 times with reagent (2).
    6) Luminescence is developed in the wells of the plate by adding 100 μl luminescence reagent (7) to each well and incubating for 20 minutes with shaking in a luminometer (Luminoscan Labsystems, Helsinki).
    7) Luminescence in relative light units for a given sample is measured and compared with that of standard type IV cPLA2 preparations. The use of standard type IV cPLA2 preparations can be used to derive a calibration curve for an assay for cPLA2. FIG. 2 shows a calibration curve derived from 10 separate assays with the standard error of the mean marked.
    8) Sample preparation
    
        Prior to analysis, the frozen red blood cells were thawed, centrifuged at 10,000 g for 5 minutes and diluted 1:40 with sample diluent (reagent (4)).
    
    
    9) Standard preparation
    
        A U937 cell cytosol was utilised as a standard. This was prepared from U937 human monocyte culture (Clark et al., Proc. Nat. Acad. Sci. USA, 87, 7708–7712), Cells were harvested from culture, resuspended in standard/sample diluent, omitting the albumin content and with the addition of 11.6 g/100 ml sucrose. The cells were disrupted by nitrogen cavitation in a Parr bomb and the resulting preparation centrifuged at 150,000 g for 1 hour at 4° C. The supernatant, a U937 cell cytosol, was aliquoted and stored at −80° C. for standardisation. Western blot analysis of dilutions of this material allowed allocation of a value for type IV cPLA2 content from the point at which type TV cPLA2 was just detected by Western blot. The standard is stable at −80° C. All assays included internal quality control samples of an aliquoted red cell haemolysate.
    
    
    10) Units for the estimation of type IV cPLA2 in red cells haemolysates are μg cPLA2 per g haemoglobin.
    
        Haemoglobin amounts were estimated by diluting the sample to 0.04% ammonia and reading the optical density at 540 mm, standardised against haemoglobin of known concentration.
        Within-batch percentage coefficient of variation (CV %), which is a measurement of relative variation, i.e. the standard deviation expressed as a percentage of the arithmetic mean, typically measured 8.6% and between-batch CV % typically measured 10.8%. These were measured at 9.0 ng cPLA2 per g of haemoglobin (ng/gHb) and 9.7 ng/gHb respectively.

IV. Measurement of Type IV cPLA2 Levels in Red Blood Cells in Various Patient Groups

    
    


As an illustration of the clinical application of the measurement in red cells of type IV cPLA2 protein or protein immunologically homologous to type IV cPLA2, various blood samples were collected from groups of patients each with a defined psychiatric illness and from a control group. The samples from each patient group in this study were analysed using the ELISA method hereinbefore described. The patients within each group met stated criteria for their clinical diagnosis, as detailed below. Samples were collected using steps 1 to 7 hereinbefore described. Specimens were also collected and prepared omitting washing steps 4 to 5. This alternative shortened cell preparation method, employing only steps 1 to 3 and 6 to 7 was examined for its influence on the assayed levels of cPLA2. Comparisons were made of samples given the full wash procedure and samples using the shortened procedure. No significant difference was found either within groups or combining the results in all groups when the two red cell preparation procedures were compared. The patients groups were as follows:
Group 1: Schizophrenia


    49 Patients who met the DSM IV criteria for the diagnosis of schizophrenia (Diagnostic and Statistical Manual of Mental Disorders 4th Edition, American Psychiatric Association 1994);
    36 Males and 13 females, age range 17 to 66 with mean age 37.3+/−11.9 years.

Group 2: Dyslexia

    27 Volunteers with recognised criteria for identification of Dyslexia, i.e. a difference of 15 points or more between Wechsler Adult Intelligence Scale Revised (WAIS-R) pro-rated equivalent IQ (© The Psychological Corporation Limited 1986) and Wide Range Achievement Test score (WRAT) (Wilkinson © Wide Range Inc, 1993) or reaching a Bangor Dyslexia Test score (T. R. Miles in Dyslexia, The Pattern of Difficulties. Grenada, 1983) equal to or greater than 7;
    17 Males and 10 females of age range 16 to 67 with mean age 35.4+/−13.2 years.

Group 3: Controls

    51 volunteers comprised the control group;
    25 Males and 26 females, age range 16 to 57 with mean age 35.4+/−12.4 years.


The results of the study are displayed in FIGS. 3A, 3B and 3C which show the frequency histogram of red cell type IV cPLA2 or proteins or proteins immunologically homologous to type IV cPLA2 in μg cPLA2 per g haemoglobin (Hb) as estimated by the ELISA method hereinbefore described. FIGS. 3A, 3B and 3C illustrate, respectively, the control group, the schizophrenic group and the dyslexic group.
The distribution of the results of the assays for red cell type IV cPLA2 or proteins or proteins immunologically homologous to type IV cPLA2 measured as μg per g haemoglobin (Hb) was positively skewed (FIG. 3) and non-parametric tests were applied in analysis. Mann Whitney U test of the schizophrenia patient group and controls reveal a highly significant elevation of red cell cPLA2 in the schizophrenia group compared with the control group (P<0.0001). There was a lesser elevation of red cell cPLA2 in the dyslexic subjects compared with controls (P<0.001). Determination of a reference range encompassing 95% of the control group for red cell cPLA2 permitted determination of an upper cut off point for the reference range for red cell cPLA2 of 2.8 μg per g Hb. This is not an absolute value but is derived from the standardisation procedure used for these studies, as hereinbefore described.
Confirmation of the ELISA analysis of the type IV cPLA2 proteins or proteins immunologically homologous to type IV cPLA2 in the red cell haemolysates may be obtained by using SDS PAGE and western blot analysis using the techniques previously described. Samples from a patient in the control group and from a patient in the schizophrenic patient group (group 1) were subjected to further analysis using SDS PAGE and western blot. The group 1 sample was taken from a patient that had been shown by ELISA to have high levels of type IV cPLA2 proteins or proteins immunologically homologous to type IV cPLA2 in the red cell haemolysates.
FIG. 4 illustrates the type IV cPLA2 proteins or proteins immunologically homologous to type IV cPLA2 in the red cell haemolysates, and the proteins observed in a red cell haemolysate from a control patient, as observed by SDS PAGE/western blot. Protein of the molecular weight of cPLA2 protein from human monocyte U937 cells is observed in the haemolysate from the schizophrenic patient; there was no protein detected which had this molecular weight in the haemolysate from the control subject. The various samples in the western blot analysis shown in FIG. 4 are as follows:

    Lane 1: Molecular weight markers
    Lane 2: Cytosol from insect cells infected with baculovirus expressing type IV cPLA2 
    Lane 3: Cytosol from human monocyte U937 cells containing a cPLA2 protein
    Lane 4: Red cell haemolysate from a control subject
    Lane 5: Red cell haemolysate from a schizophrenia subject

## Claims

1. An assay for detecting type IV cytosolic phospholipase A2 (cPLA2) protein or a protein immunologically homologous to type IV cPLA2, the assay comprising
i) obtaining a sample of red blood cells separated from whole blood, and
ii) detecting said type IV cytosolic phospholipase A2 (cPLA2) protein or said protein immunologically homologous thereto in or on said red blood cells with an antibody to type IV cPLA2 protein.

2. An assay according to claim 1 for use in the diagnosis of a disease in which dysfunction of cell signalling systems involving fatty acids having three or more carbon—carbon double bonds is implicated.

3. An assay according to claim 2 wherein said disease is a disease or disease process in which type IV cPLA2 activity or concentration is altered from normal levels.

4. An assay according to claim 2 wherein said disease is a disease or disease process in which type IV cPLA2 activity or concentration is increased relative to normal levels.

5. An assay according to claim 2 wherein the disease is schizophrenia, dyslexia, bipolar or manic depressive illness, cachexia or brain injury.

6. An assay according to claim 5 wherein the brain injury is stroke or mechanical brain injury.

7. An assay according to claim 1 for use in monitoring the effectiveness of medication administered to a patient suffering from a disease in which dysfunction of cell signalling systems involving fatty acids having three or more carbon—carbon double bonds is implicated.

8. An assay according to claim 1 for use in drug development for a disease in which dysfunction of cell signalling systems involving fatty acids having three or more carbon—carbon double bonds is implicated.

9. An assay according to claim 1 wherein the red blood cells are isolated from the human body.

10. An assay according to claim 1 wherein the type IV cPLA2 protein or the protein immunologically homologous to type IV cPLA2 has a molecular weight in the range 80 to 110 kDa or in the range 70 to 80 kDa or in the range 50 to 60 kDa.

11. An assay according to claim 1 wherein the type IV cPLA2 protein or the protein immunologically homologous to type IV cPLA2 has a molecular weight in the range 90 to 105 kDa or in the range 70 to 80 kDa or in the range 50 to 60 kDa.

12. An assay according to claim 1 wherein said proteins are detected using an antibody or antibodies raised against an epitope or epitopes from amino acids 1 to 216 of type IV cPLA2 protein from human monocyte cells.

13. An assay according to claim 1 wherein said proteins are detected using an antibody or antibodies that recognise an epitope or epitopes from amino acids 82 to 749 of type IV cPLA2 protein from human monocyte cells.

14. An assay according to claim 13, wherein two or more of the antibodies are used in combination or in sequence to detect the said proteins with the required specificity.

15. An assay according to claim 1 wherein said proteins are detected using an antibody or antibodies raised against an epitope or epitopes from amino acids 82 to 749 of type IV cPLA2 protein from human monocyte or raised against an epitope or epitopes of a synthetic peptide matching amino acids 82 to 749 of type IV cPLA2 protein from human monocyte cells.

16. An assay according to claim 13 or 15 wherein said epitope or epitopes are from a peptide sequence or sequences which comprise the catalytic centre of type IV cPLA2 protein from human monocyte cells.

17. An assay according to claim 13 or 15 wherein said epitope or epitopes are from the peptide sequence of amino acids 241 to 260 of type IV cPLA2 protein from human monocyte (U937) cells.

18. An assay according to claim 13 or 15 wherein the human monocyte cells are of the (U937) cell line.

19. A method of diagnosis of a disease in which dysfunction of cell signalling systems involving fatty acids having three or more carbon—carbon double bonds is implicated, said method comprising:
i) obtaining a sample of red blood cells separated from whole blood, and
ii) detecting type IV cytosolic phospholipase A2 (cPLA2) protein or a protein immunologically homologous to type IV cPLA2 in or on red blood cells with an antibody to type IV cPLA2 protein.

20. A method of monitoring the effectiveness of medication administered to a patient suffering from a disease in which dysfunction of cell signalling systems involving fatty acids having three or more carbon—carbon double bonds is implicated, said method comprising:
i) obtaining a sample of red blood cells separated from whole blood, and
ii) detecting type IV cytosolic phospholipase A2 (cPLA2) protein or a protein immunologically homologous to type IV cPLA2 in or on red blood cells with an antibody to type IV cPLA2 protein.

21. A method of drug development for a disease in which dysfunction of cell signalling systems involving fatty acids having three or more carbon—carbon double bonds is implicated, said method comprising:
i) obtaining a sample of red blood cells separated from whole blood, and
ii) detecting type IV cytosolic phospholipase A2 (cPLA2) protein or a protein immunologically homologous to type IV cPLA2 in or on red blood cells with an antibody to type IV cPLA2 protein.

22. An assay for detecting type IV cytosolic phospholipase A2 (cPLA2) protein or a protein immunologically homologous to type IV cPLA2 comprising the steps of:
i) collecting a sample of blood from a subject, and
ii) detecting the proteins.

23. An assay according to claim 22 further comprising one or more of the following steps after obtaining the sample of red blood cells and prior to detecting the proteins:
(a) separating the red blood cells from other blood components,
(b) disrupting the red blood cells,
(c) separating the proteins using a protein separation technique.

24. An assay according to claim 23 wherein the red cells are disrupted by sonication, freezing, nitrogen cavitation or lysis.