PROTEIN P5, A SERUM MARKER FOR BRAIN DAMAGE

Abstract: Protein P5 is a blood or serum marker for damage to brain or the blood brain barrier. The present invention is directed to the use of an assay for Protein P5 to detect damage to the brain or the blood brain barrier.

Description: PROTEIN P5, A SERUM MARKER FOR BRAIN DAMAGE Background of the Invention Field of the Invention The invention relates to the identification of protein P5 in blood or serum as an indicator of brain or blood brain barrier damage. The invention also relates to a method of diagnosing brain or blood brain barrier damage by determining the presence or amount of the protein P5 in blood or serum. Description of Related Art The brain and its surrounding fluid, the spinal fluid, are separated from the blood compartment by a barrier called the blood-brain barrier. Transport between the blood and the spinal fluid is thought to be primarily limited by specialized tight junctions in the basement membrane between plasma and brain, that create a special blood-brain barrier. In diseases of the meninges or of the brain, this blood-brain barrier can be disrupted and proteins from the blood can leak into the spinal fluid. It is not clear whether the reverse is true, i.e., whether brain or spinal fluid specific proteins might leak into the blood as well. If so, then it should be possible to manufacture a blood test for brain or meninges diseases by using highly specific monoclonal antibodies against a protein that is normally found only in the brain or spinal fluid. Harrington et al (1993) , and others, have described a spinal fluid protein called protein P5 which is an abundant protein on two dimensional electrophoresis gels of spinal fluid protein. P5 is a beta-trace protein which appears to be specific to the brain and spinal fluid. Harrington et al described the purification method, the sequencing of P5 fragments, the production of polyclonal antibodies against synthetic peptides derived from P5 sequences, and the study of numerous organs and body fluids with the produced polyclonal antibodies. The antibodies were not specific; eight other proteins were detected which reacted with the antibodies but have different isoelectric points and molecular weights. The studies of numerous organs and body fluids indicate that P5 is found only in spinal fluid and brain. The function as well as the detailed structure of the group of beta-trace proteins, of which P5 appears to be one, is not entirely understood. The absence of beta- trace proteins from other tissues indicates a high tissue specificity. Such a prominent central nervous system protein presumably reflects a localized function. It has been proposed that P5 could be involved in a neuro- biological cell surface role such as cell adhesion molecules. The amino acid sequences of the protein has short regions of similarity of five sequence fragments with the intermediate filament lamin proteins (Fisher et al, 1986) , cell-to-cell adhesion molecules, cadhedrin (Hatta et al, 1988) and contactin (Ranscht and Dours, 1988) . Therefore, cell adhesion or some similar structural or biochemical function has been proposed for P5. Previous reports indicated that beta-trace protein is the same peptide as prostaglandin D synthetase (Zahn et al, 1993) . The present inventor has sequenced the protein and confirmed that P5 is identical to prostaglandin D synthetase. The presence or absence of the protein has also been studied in various diseases of the nervous system. The results of assaying P5 in cerebrospinal fluid from patients has been either conflicting or negative. Patients with diseases of the nervous system showed inconsistent or no changes from normal CF (Link & Olsson, 1972) . In some patients with Creutzfeldt-Jakob disease, some abnormalities on a two dimensional gel electrophoresis of spinal fluid are seen. To date however, protein P5 has not been confirmed as an indicator of any particular condition or disease. SUMMARY OF THE INVENTION The inventors have discovered that the presence or amount of P5 in the blood is an indicator of meningitis, stroke, and other brain or blood-brain barrier damage. The inventors have devised assays by which the presence of P5 in blood or serum may be determined. Such assays include immunoassays, such as ELISA, one and two dimensional gel electrophoresis, and one and two dimensional gel electrophoresis-immunostain blots. The most preferred test is an immunoassay, such as an ELISA of blood or serum, or an immunoPCR method, using monoclonal antibodies against the full native P5 peptide. BRIEF DESCRIPTION OF THE DRAWING The figure shows a two dimensional electrophoresis gel of spinal fluid. The position of one of the isoforms of the peptide P5 is marked. DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1 - isolation of the protein. Protein P5 is present in large amounts in the spinal fluid. This glycoprotein has a molecular weight range of 18,000 - 24,000 daltons. It displays several isoelectric points due to its icroheterogeneity. The protein may be isolated from spinal fluid in a variety of ways. In one method, spinal fluid obtained from a healthy volunteer is affinity purified by passing the fluid over a Sepharose column with antiserum against pooled serum proteins attached via cyanogen bromide (Pharmacia 17- 0430-01) . P5 enriched spinal fluid that did not attach to the column can then be concentrated by ultrafiltration and the filtrate may be run on two dimensional (2D) electrophoresis gels. The preferred gels are 2D Immobilized pH Gradient gels (IPG) . The P5 spot may be identified and the corresponding sections cut out of the gels and the peptide eluted. Alternatively, the P5 enriched fraction from the affinity chromatography may be run over a second affinity column which is a Sepharose column conjugated with polyclonal or monoclonal antibodies against Protein P5. Other purification procedures known to those skilled in the art of protein purification may also be used. The peptides may also be made by recombinant means, or by chemical synthesis. In a preferred embodiment, P5 is produced by using conventional genetic engineering techniques using the cDNA gene of P5 expressed in E. coli . . The cDNA is taught in Nagata et al, 1991. Various systems for expression of heterologous genes are well known to those skilled in the art. A polyhistidine tail added to the recombinant protein provides for a means to efficiently purify the recombinant P5. Example 2 - characterization of the protein. The blotting of proteins separated by two- dimensional polyacrylamide gel electrophoresis (PAGE) onto polyvinylidene difluoride (PVDF) membranes (Towbin et al, 1979; Matsudaira 1987; Eckerskorn et al, 1988; and Eckerskorn et al 1990) has enabled the identification and characterization of proteins from complex biological samples . Transfer of the proteins can be carried out using several methods such as vacuum, capillary or electric field. Electroblotting is by far the most wide¬ spread technique which utilizes either vertical buffer tanks or semi-dry blotting. Both techniques can use either the Towbin or 3- [cyclohexamino] -1-propanesulfonic acid (CAPS) transfer buffers, depending on the need for minimal glycine contamination in post-transfer protein characterization. These two buffer systems are described below. Gloves must be worn and all filter papers should be washed three times for 3 min in transfer buffer. These two steps are important in order to avoid any protein or amino acid contamination. 1. Towbin buffer svstem (a) After second-dimensional electrophoresis, soak the gels in deionized water for 3 min. (b) Equilibrate the gels in a solution containing Tris (13mM) , glycine (lOOmM) and methanol (10% v/v) for 30 min. At the same time, wet PVDF membranes in methanol for 1 min and equilibrate them in a solution containing Tris (13mM) , glycine (100 mM) and methanol (10% v/v) also for 30 min. (c) Carry out electroblotting either in: i. a transfer tank with a solution containing Tris (13mM) , glycine (lOOmM) and methanol (10% v/v) at 90 V constant voltage for 3 hours at 15°C. Assemble the blotting sandwich. ii. or a semi-dry apparatus with a solution containing 10 mM CAPS pH 11 and methanol (20% v/v anodic side; 5% v/v cathodic side) at 1 mA/cm 2 constant current for 3 hours at 15°C or as described by the manufacturer. 2. CAPS buffer system (a) After second-dimensional electrophoresis, soak the gels in deionized water for 3 min. (b) Equilibrate the gels in a solution containing 10 mM CAPS pH 11 for 30 min. At the same time, wet PVDF membranes in methanol for 1 min and equilibrate them in a solution containing 10 mM CAPS pH 11 and methanol (10% v/v) also for 30 min. (c) Carry out electroblotting in either: i. a transfer tank with a solution containing 10 mM CAPS pH 11 and methanol (10% v/v) at 90 V constant voltage for 3 hours at 15°C. Assemble the blotting sandwich. ii. or a semi-dry apparatus with a solution containing 10 mM CAPS pH 11 and methanol (20% v/v anodic side; 5% v/v cathodic side) at 1 mA/cm 2 constant current for 3 hours at 15°C or as described by the manufacturer. 2-D PAGE and electroblotting onto PVDF membranes have become widely used techniques for the characterization of proteins. The improvements which allowed a higher protein load and a better transfer yield have also assisted accurate protein microsequencing and amino acid composition analysis. However, the application of these techniques to proteins would not have been possible without the development of complementary detection methods. Amido Black, Coomassie Brilliant Blue R-250, colloidal gold and Ponceau S are commonly utilized to visualize proteins on PVDF membranes (see below) and are compatible with the ensuing protein identification chemistry (Sanchez et al, 1992) . However, the use of amido black staining is preferred if membranes are to be used for AA analysis. Protein stains for PVDF membranes 1. Amido Black (a) After electrotransfer, stain the PVDF membranes in a solution containing Amido Black (0.5% w/v) , isopropanol (25% v/v) and acetic acid (10% v/v) for 1 min. (b) Destain by several soakings in deionized water on a shaker. 2. Coomassie Brilliant Blue R-250 (a) After electrotransfer, stain the PVDF membranes in a solution containing Coomassie Brilliant Blue R-250 (0.1% w/v) and methanol (50% v/v) for 15 min. (b) Destain in a solution containing methanol (40% v/v) and acetic acid (10% v/v) . 3. Colloidal Gold (Progold) (a) After electrotransfer, incubate the PVDF membranes in phosphate-buffered saline (PBS) pH 7.2 with Tween 20 (0.5% v/v) for 30 min. (b) Wash 3x5 min in PBS-Tween 20 (0.5% v/v) and 1 min. in deionized water. (c) Stain the membranes overnight at room temperature in 100 ml of a solution containing colloidal gold such as Progold™ or Aurodye™. 4. Ponceau S (a) After electrotransfer, stain the PVDF membranes in a solution containing Ponceau S (0.2% w/v) and TCA (3% v/v) . (b) Destain by several soakings in deionized water. Drying and scanning Dry the stained PVDF membranes on a glass plate of 3 mm thickness at 37°C for 10 min. The membranes can then be scanned on a laser densitometer and analyzed with image analysis software such as Melanie II. Edman degradation identification Automated Edman degradation, which produces N- terminal protein sequence, is a common identification method for PVDF-bound proteins (Matsudaira, 1987) . Sequencing is usually done for 15 cycles, and identity is established by matching the sequence obtained against those in protein databases . Internal protein sequencing is also possible, but is a more labor-intensive procedure. The present inventor has sequenced the P5 protein by the above methods and confirmed that P5 is identical to prostaglandin D synthetase. Example 3 - Preparation of antibodies The protein obtained in Example 1 is then used to prepare monoclonal antibodies which are highly specific for protein P5. (Fragments of the P5 peptide may also be used as immunogen.) If fragments are used it may be necessary to conjugate the fragment to an immunogen to increase the immune response. Monoclonal antibodies may be prepared by procedures well known in the art, such as that of Kohler and Milstein (1975) . In a preferred embodiment the monoclonal antibodies are produced in a mouse model. The primary cultures may be cloned in the usual way, i.e, using commercial cell sorters or by limiting dilution. The clones so obtained are then tested to determine that the antibodies they produce react with protein P5, and do not react with other spinal fluid or blood proteins. The cell lines obtained are also tested to determine if the antibodies which they produce bind to the same epitope of the protein or if they bind to different epitopes. Specific monoclonals which bind selective epitopes are screened to identify P5 brain specific glycosylation or posttranslational modifications. Example 4 - Immunoassays The monoclonal antibodies obtained from the hybridoma cell lines generated in Example 3 may then be used in an immunoassay to determine the presence or amount of protein P5 in blood or serum. All known immunoassays are suitable for use in this method. The assays may be competitive assays, sandwich assays, and the label may be selected from the group of well known labels such as radioimmunoassay, enzymeimmuno assay, fluorescent or chemiluminescence immunoassay, or immunoPCR technology for further sensitivity enhancement. Immuno-PCR is taught in Hendrickson et al, 1995. The particularly preferred embodiment of the present invention is an ELISA. In such an assay, a body fluid of the patient, preferably blood or serum, is tested to determine the presence or amount of P5 protein. The presence of P5 protein in a body fluid other than spinal fluid, such as serum or plasma, is taken as an indication that the patient has suffered some kind of brain or blood brain barrier damage, for instance, stroke. In some instances, it may be preferable to subject the body fluid specimen to one or two dimensional gel electrophoresis, and then either do standard protein stains, or immunostains with one of the antibodies of the invention. The peptide is identified by its mobility compared to known P5 peptide. An aliquot of P5 from CSF is added to a blood or serum sample and run on an 2D electrophoresis gel to produce a reference gel which shows the position of P5 compared to blood and serum proteins. The blood test for meninges disease of the present invention is surprising in that it is the first blood test available for diseases of the brain, blood brain barrier, or meninges. The assays of the present invention such as ELISA, or agglutination tests are rapid and can provide much needed data in the care of patients who are suspected of having suffered brain, blood brain barrier or meninges damage. REFERENCES The following publications are hereby incorporated by reference. Eckerskorn, C, Jungblut, P., Mewes, W. , Klose, J. and Lottspeich, F. (1988) . Electrophoresis, 9, 830-838. Eckerskorn, C. and Lottspeich, F. (1990) , Electrophoresis, 11, 554-561. Fisher et al, Proc. Natl . Acad. Sci . 83:6450-6454, (1986) Harrington et al, Applied and Theoretical Electrophoresis, 3:229-234, (1993) . Hatta et al, J " . Cell Biol. 106:873-881, (1988) . Hendrickson et al, Nucleic Acids Research, 23 (3) .-522-529 (1995) . Kohler and Milstein, Nature 256:495 - 497 (1975) . Link & Olsson, Acta. Neurol. Scand. 43:7-136, (1972) . Matsudaira, P. (1987) . J. Biol. Chem., 262, 10035-10038. Nagata et al, Proc. Natl. Acad. Sci. U.S.A., 88:4020-4024 (1991) . Ranscht and Dours, J. Cell Biol. 107:1561-1573, (1988) . Sanchez, J.-C, Ravier, F., Pasquali, C, Frutiger, S., Paquet, N. , Bjellqvist, B., Hochstrasser, D.F. and Hughes, F.J. (1992) . Electrophoresis , 13, 715-717. Towbin, H., Staehelin, T. and Gordon, J. (1979) . Proc. Natl. Acad. Sci. USA, 76, 4350-4354. Zahn et al, Journal of Neurochemistry, 61 (2) :451-456, (1993) .

Claims: CLAIMS 1. Use of the protein P5 or an antibody thereto in the diagnosis from a blood or serum sample, of damage to the brain or the blood-brain barrier. 2. A method of detecting damage to the brain or the blood brain barrier comprising: a) performing an assay on the blood or serum of a patient who is suspected of having suffered damage to the brain or blood brain barrier; b) determining the presence or amount of P5; c) correlating the presence or amount of P5 with the occurrence of damage to the brain or the blood brain barrier. 3. The method of claim 2 which is an immunoassay. 4. The method of claim 3, wherein the immunoassay is an ELISA. 5. The method of claim 3, wherein the immunoassay is an agglutination assay. 6. The method of claim 3, wherein the assay comprises carrying out a one or two dimensional gel electrophoresis on the blood or serum sample, thus separating P5, immunoblotting P5 with an antibody thereto and detecting the immunoblot. 7. The method of claim 3, wherein the immunoassay is an immuno-polymerase chain reaction assay. 8. A monoclonal antibody which specifically binds protein P5, and does not cross-react with any other protein of the spinal fluid or blood. 9. A hybridoma cell line which produces a monoclonal antibody which specifically binds protein P5, and does not cross-react with any other protein of the spinal fluid or blood.

Inventors: HOCHSTRASSER DENIS, HOCHSTRASSER, DENIS
Applicants: ELECTROPHORETICS INTERNATIONAL, ELECTROPHORETICS INTERNATIONAL PLC
Application year: 1996
Main classification: G01N33
IPC codes: C07K16, C12N5, C12N9, C12N15, C12P21, C12Q1, C12R1, G01N27, G01N33