Heart and brain fatty acid binding proteins (H-FABP, B-FABP) are markers for stroke. The invention provides a diagnostic assay for either of these markers, preferably by ELISA using anti-H-FABP or B-FABP antibody. Since H-FABP is also a marker for acute myocardial infarction (AMI), to distinguish stroke from AMI requires an assay specific to AMI, e.g. using troponin-I or creatine kinase-MB as a marker, also to be carried out.

SUMMARY OF THE INVENTION

It has now surprisingly been found that two fatty acid binding proteins (FABP), known as heart (H-FABP) and brain (B-FABP), are markers for stroke. Thus, the invention provides a method of diagnostic assay for stroke or the possibility thereof in a sample of body fluid taken from a patient suspected of suffering from a stroke, which comprises determining the concentration of heart or brain fatty acid binding protein (H-FABP or B-FABP) in the sample. The concentration thus determined is used to make or assist in making a diagnosis.

Conveniently the method is carried out using an antibody to H-FABP or B-FABP, whereby the extent of the reaction between the antibody and the FABP in the sample is assayed and related to the concentration of FABP in the sample.

The present invention enables an assay of high sensitivity, specificity and predictive positive value for stroke to be carried out. "Sensitivity" is defined as the percentage of true positives given by the assay on samples taken from patients in whom clinical examination has confirmed stroke. It is reckoned as % True positives/(True positives+False negatives). "Specificity" means the percentage of true negatives given by the assay on control samples, i.e. from patients in whom clinical examination has not revealed stroke. It is reckoned as % True negatives/(False positives+True negatives). "Predictive positive value" means the ratio % True positives/(True positives +False positives).

H-FABP is a known marker of acute myocardial infarction (AMI), see J. Ishii et al., "Serum concentrations of myoglobin vs human heart-type cytoplasmic fatty-acid binding protein in early detection of acute myocardial infarction", Clinical Chemistry 1997;43 1372 1378. Therefore, in order to use an assay for H-FABP for stroke to better advantage, it is desirable to perform another kind of assay for AMI (one in which the marker is not a FABP) in order to eliminate from the diagnosis for stroke those patients who are positive in the AMI assay.

Thus, in a particular embodiment, the invention provides a method which comprises determining the concentration of H-FABP in a first assay, as defined above, whereby a positive result indicates the possibility of either a stroke or acute myocardial infarction, and which further comprises carrying out a second diagnostic assay, for acute myocardial infarction (AMI) only, whereby a positive result in the H-FABP assay and a negative result in the assay for AMI indicates that the patient might be suffering from a stroke. Assays using Troponin-I and Creatine Kinase-MB (CK-MB) as early biochemical markers of acute myocardial infarction (AMI) are well known and suitable for the above purpose. They can be carried out in plasma, serum or blood. Of course, the terms "first" and "second" are merely convenient labels: the two assays can be carried out in either order.

A similar H-FABP and also a brain-specific fatty acid binding protein (B-FABP) have been found in the brain of mice, see L. Pu et al., Molecular and Cellular Biochemistry 1999;198 69 78. Brain H-FABP (not to be confused with B-FABP) is believed to differ from heart H-FABP by a single amino acid substitution. However, B-FABP differs considerably. P. A. Sellner et al., "Development role of fatty acid binding proteins in mouse brain" Dev. Brain Res. 1995;89:33 46 estimated the DNA homology at 69%, while A. Schreiber et al., "Recombinant human heart-type fatty acid binding protein as standard in immunochemical assays" mention 64% amino acid sequence homology and that a monoclonal antibody to human H-FABP is cross-reactive with human B-FABP to the extent of only 1.7%.

Now that the present inventors have found that H-FABP is a marker for stroke, it is a very reasonable prediction that B-FABP will also be. Since B-FABP is specific to brain tissue and does not appear to react significantly with a monoclonal antibody to H-FABP, it will not give positives for AMI, making a separate assay for AMI unnecessary.

DESCRIPTION OF PREFERRED EMBODIMENTS

For the method of assay, the sample can be taken from the blood, plasma or serum of the patient. The marker, H-FABP or B-FABP, is preferably measured by an immunoassay, using a specific antibody to H-FABP and measuring the extent of the antigen (H-FABP or B-FABP)/antibody interaction. For the diagnosis of human patients, the antibody is preferably anti-human H-FABP or B-FABP. Similarly, if the patient is an animal the antibody should be to the H-FABP or B-FABP of the same animal variety, e.g. anti-equine H-FABP or B-FABP if the patient is a horse. It may be a monoclonal antibody or an engineered antibody. Conveniently a mouse anti-human, anti-equine etc. monoclonal antibody is used. Antibodies to H-FABP are known, e.g. 66E2 and 67D3 described by W. Roos et al., "Monoclonal antibodies to human heart type fatty acid-binding protein", J. Immunol. Methods 1995;183 149 153, are commercially available. Also, the usual Kohler-Milstein method may be used to raise H-FABP or B-FABP antibodies. The source of protein for this purpose can be the naturally derived or recombinant DNA-prepared protein. Recombinant human H-FABP and B-FABP have been described by A. Schreiber supra and F. Shimizu et al., "Isolation and expression of a cDNA for human brain fatty acid binding protein (B-FABP)", Biochim. Biophys. Acta 1997;1354:24 28, respectively. Less preferably, the antibody may be polyclonal.

Any known method of immunoassay may be used. A sandwich assay is preferred. In this method, an antibody (e.g. polyclonal) to the FABP is bound to the solid phase such as a well of a plastics microtitre plate, and incubated with the sample and with a labelled second antibody specific to the H-FABP or B-FABP to be detected. Alternatively, an antibody capture assay (also called "indirect immunoassay") could be used. Here, the test sample is allowed to bind to a solid phase, and the anti-FABP antibody (polyclonal or monoclonal) is then added and allowed to bind. If a polyclonal antibody is used in this context, it should desirably be one which exhibits a low cross-reactivity with other forms of FABP. After washing away unbound material, the amount of antibody bound to the solid phase is determined using a labelled second antibody, anti- to the first.

A direct assay could be performed by using a labelled anti-FABP antibody. The test sample is allowed to bind to the solid phase and the anti-FABP antibody is added. After washing away unbound material, the amount of antibody bound to the solid phase is determined. The antibody can be labelled directly rather than via a second antibody.

In another embodiment, a competition assay could be performed between the sample and a labelled FABP or a peptide derived therefrom, these two antigens being in competition for a limited amount of anti-FABP antibody bound to a solid support. The labelled FABP or peptide could be pre-incubated with the antibody on the solid phase, whereby the FABP in the sample displaces part of the FABP or peptide thereof bound to the antibody.

In yet another embodiment, the two antigens are allowed to compete in a single co-incubation with the antibody. After removal of unbound antigen from the support by washing, the amount of label attached to the support is determined and the amount of protein in the sample is measured by reference to standard titration curves established previously.

Throughout, the label is preferably an enzyme. The substrate for the enzyme may be colour-forming, fluorescent or chemiluminescent. Alternatively, the label may be a radioisotope or fluorescent, e.g. using conjugated fluorescein.

The enzyme is preferably alkaline phosphatase or horseradish peroxidase and can conveniently be used colorimetrically, e.g. using p-nitrophenyl phosphate as a yellow-forming substrate with alkaline phosphatase.

For a chemiluminescent assay, the antibody can be labelled with an acridinium ester or horseradish peroxidase. The latter is used in enhanced chemiluminescent (ECL) assay. Here, the antibody, labelled with horseradish peroxidase, participates in a chemiluminescent reaction with luminol, a peroxide substrate and a compound which enhances the intensity and duration of the emitted light, typically 4-iodophenol or 4-hydroxycinnamic acid.

An amplified immunoassay such as immuno-PCR can be used. In this technique, the antibody is covalently linked to a molecule of arbitrary DNA comprising PCR primers, whereby the DNA with the antibody attached to it is amplified by the polymerase chain reaction. See E. R. Hendrickson et al., Nucleic Acids Research 1995; 23, 22 529 (1995) or T. Sano et al., in "Molecular Biology and Biotechnology" ed. Robert A. Meyers, VCH Publishers, Inc. (1995), pages 458 460. The signal is read out as before.

In a particularly preferred procedure, an enzyme-linked immunosorbent assay (ELISA) was developed to detect H-FABP. Since H-FABP is a marker for AMI as well, Troponin-I or CK-MB concentrations were assayed in order to exclude any heart damage. As described in the Example, These assays were assessed in serial plasma samples, from 22 patients lacking AMI and stroke, 20 patients with AMI and 22 patients with confirmed stroke at four times points after the admission at the medical centre. The sensitivity, specificity and predictive positive value for H-FABP in stroke were 59.1%, 90.9% and 86.7% respectively. Only one out of 22 stroke patients had increased H-FABP and Troponin-I expression. Thus, H-FABP detection combined with the Troponin-I or CK-MB assay provide a useful marker of stroke diagnosis or brain damage.

The use of a rapid microparticle-enhanced turbidimetric immunoassays, developed for H-FABP in the case of AMI, M. Robers et al., "Development of a rapid microparticle-enhanced turbidimetric immunoassay for plasma fatty acid-binding protein, an early marker of acute myocardial infarction", Clin. Chem. 1998;44:1564 1567, should drastically decrease the time of the assay. Thus, the full automation in a widely used clinical chemistry analyser such as the COBAS.TM. MIRA Plus system from Hoffmann-La Roche, described by M. Robers et al. supra, or the AxSYM.TM. system from Abbott Laboratories, should be possible and applied for routine clinical diagnosis of stroke.

The H-FABP or B-FABP concentrations can be measured by other means than immunoassay. For example, the sample can be subjected to 2D-gel electrophoresis and the amount of the FABP estimated by densitometric scanning of the gel or of a blot therefrom. However, it is desirable to carry out the assay in a rapid manner, so that the patient can be treated promptly.

 

Claim 1 of 7 Claims

1. A diagnostic assay for stroke comprising obtaining a body fluid sample from a subject; measuring the sample from the subject for heart fatty acid binding protein and for a marker of acute myocardial infarction; comparing the level of heart fatty acid binding protein and the level of the marker of acute myocardial infarction measured in the sample from the subject with the level of heart fatty acid binding protein and the level of the marker of acute myocardial infarction in a control sample obtained from subjects not suffering from stroke and acute myocardial infarction, wherein elevated levels of heart fatty acid binding protein, in combination with non-elevated levels of the marker of acute myocardial infarction, in the sample from the subject compared with the control sample, indicates that the subject is suffering from a stroke.
 

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