ES2657320T3 - Diagnostic method for disorders related to brain damage - Google Patents

Abstract

A method of diagnosing a disorder related to brain damage selected from head trauma, ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, intracranial hemorrhage, transient ischemic attack and vascular dementia or the possibility of them, in a subject suspected of suffering the same, or of prognosis or therapeutic follow-up of said disorder related to brain damage in a subject, which comprises detecting H-FABP and at least one additional polypeptide selected from the RNA-binding regulatory subunit, homolog of degradation protein 1 by fusion of ubiquitin and nucleoside diphosphate kinase A, or respective variants or mutants thereof, which has at least 90% homology thereto and which has substantially the same functional and immunological properties, in a sample of body fluid taken from the subject , wherein said H-FABP and at least one additional polypeptide or said variant or mutant, is n present in the body fluid of subjects affected by a disorder related to brain damage in a greater amount compared to the body fluid of subjects affected by a disorder not related to brain damage, so that a greater amount of said H- FABP and at least one additional polypeptide or said variant or mutant in the body fluid sample is indicative of said disorder related to brain damage.

Description

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Peroxirredoxin family in brains of patients with different neurodegenerative diseases (Krapfenbauer et al., Electrophoresis, 2002; Krapfenbauer et al., Brain Res., 2003).

(3) Peptidyl-prolyl cis-trans isomerase A or Cyclophilin A (P05092)

5 Two points were identified as Peptidyl-prolyl cis-trans isomerase A (Figure 5). This is an extension of 2-DE maps of healthy CSF and CSF of deceased prepared in the same way as in Figure 4. The basic point corresponding to Cyclophilin A is just adjacent to the point corresponding to Peroxirredoxin 5.

10 Cyclophilin A was described as a protective factor against cellular oxidative stress (Doyle et al., Biochem J., 1999). It binds to Peroxirredoxin enzymes and could be implicated in peroxidase activity (Lee et al., J. Biol. Chem., 2001). In addition, one publication suggests that cyclophilin A is secreted by vascular smooth muscle cells (VSMC) in response to oxidative stress and stimulates the growth of VSMC (Jin et al., Circ. Res., 2000). These results suggest the involvement of cyclophilin A in vascular disease processes. I also know

15 described a link with a familiar form of amyotrophic lateral sclerosis (a neurodegenerative pathology) associated with a mutation in the antioxidant enzyme Cu / Zn Superoxide Dismutase-1 (Lee at al., PNAS, 1999). Cyclophilin A appears to have a protective effect against mutant SOD-induced apoptosis.

EXAMPLE 4

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Introduction

A study of stroke patients was performed and the results are shown in Figures 6 to 15. An ELISA intensity signal was obtained for the homologue of ubiquitin fusion degradation protein 1 (UFD1), the

25 regulatory RNA-binding subunit (RNA-BP) and nucleotide diphosphate kinase A (NDK A) in plasma samples from patients and from negative control patients. Plasma samples were taken from patients between 0-24 hours and / or after 72 hours after arrival at the emergency hospital, and were compared by age / sex with samples from control patients.

30 Protocol

An ELISA was performed using black 96-well NeutrAvidin ™ coated plates Reacti-Bind ™ (Pierce, Rockford, IL). The plates were first rinsed in a borate buffer saline solution at pH 8.4 (BBS) (100 mM H3BO3, 25 mM Na2B4O7 (Sigma, St Louis, MO, United States), 75 mM NaCl (Merck, Darmastadt, 35 Germany)) in a NOVAPATH ™ washer (Bio-Rad, Hercules, CA). Then, 50 µl of biotin-conjugated antibody (2 µg / ml) prepared in dilution buffer A at pH 7 (DB, 80% polyvinyl alcohol, Mol. Weight 9000-10,000 (Aldrich, Milwaukee, WI) was added , United States), MOPS (3- [N-morpholino] propane sulfonic acid) (Sigma), NaCl, MgCl2 (Sigma), ZnCl2 (Aldrich), pH 6.90, 30% BSA solution, manufacturing grade ( Serological Proteins Inc., Kankakee, IL)) and incubated for one hour at 37 ° C. Then, the plates were washed 3 40 times in BBS in the plate washer. Then, 50 µl of antigen was added and incubated for one hour at 37 ° C. Recombinant proteins were diluted to 100, 50, 25, 12.5, 6.25 ng / ml in dilution buffer A to establish a calibration curve. Plasma samples were diluted to the appropriate dilution in dilution buffer A. After the washing step, 50 µl of antibodies conjugated with alkaline phosphatase were added to the appropriate dilution in dilution buffer A and incubated for one hour. at 37 ° C. The 96-well plate is

45 then washed 3 times with BBS in the plate washer and 50 µl of Attophos® AP fluorescence fluorescent substrate (Promega, Madison, WI) was added. The plates were immediately read on a GEMINI-XS fluorometric microtiter plate reader (Molecular Devices Corporation, Sunnyvale, California, United States) in relative fluorescence units (RFU) (λ excitation = 444 nm and λ emission = 555 nm).

The plates are read in fluorescence using a SpectraMax GEMINI-XS (Molecular Devices) fluorimetric microplate reader (λexcitation = 444 nm and λemission = 555 nm). The results are expressed in RFU and can be obtained in endpoint mode (only one reading) or in kinetic mode in 10 minutes. In kinetic mode, 6 flashes were used for each well (per well) that are integrated in an average and each well is read 6 times using a minimum time interval between each reading. This ends up being 2 minutes between readings. One was determined

55 pending and this is what was used for these assessments. The best cut-off value to discriminate between the Control and Stroke groups (ischemic plus hemorrhagic or ischemic versus hemorrhagic) was determined by ROC curves using GraphPad Prism 4 software.

conclusion

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3,125, 1.56 ng / ml in dilution buffer A to establish a calibration curve. Plasma samples were diluted to the appropriate dilution in dilution buffer A. After an additional wash step, 50 µl of antibodies conjugated with alkaline phosphatase specific to the relevant biomarker were added to the appropriate dilution in dilution buffer A and they were incubated for one hour at 37 ° C. The

5 96-well plate was then washed 3 times with BBS in the plate washer and 50 µl of fluorescent Attophos® AP fluorescent substrate (Promega, Madison, WI) was added. The plates were immediately read on a SpectraMax GEMINI-XS fluorometric microtiter plate reader (Molecular Devices Corporation, Sunnyvale, CA, United States).

10 Plates are read in fluorescence using a SpectraMax GEMINI-XS (Molecular Devices) fluorimetric microplate reader (λexcitation = 444 nm and λemission = 555 nm). The results are expressed in RFU and can be obtained in endpoint mode (only one reading) or in kinetic mode in 10 minutes. In kinetic mode, 6 flashes were used for each well (per well) that are integrated in an average and each well is read 6 times using a minimum time interval between each reading. This ends up being 2 minutes between readings. A slope was determined and

15 This is what was used for these assessments. The best cut-off value to discriminate between the Control and Ictus groups was determined by ROC curves using GraphPad Prism 4 software.

The results are shown in Figure 20.

20 EXAMPLE 7

This corresponds to Example 6, except that the polypeptide is RNA-BP. Figure 21 shows the levels of RNA-BP in the CSF of a control patient and a deceased patient. Figure 22 shows the plasma concentration of RNA-BP by ELISA for three studies, each of which includes stroke patients and controls.

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EXAMPLE 8

This corresponds to Example 6, except that the polypeptide is NDKA. Figure 23 shows the NDKA levels in the CSF of a control patient and a deceased patient. Figure 24 shows the plasma concentration of NDKA

30 by ELISA for cohorts of stroke patients and controls in Geneva and the United States as in Example 6.

EXAMPLE 9

35 In addition to simple discrimination between stroke and control patients, the data from each of Examples 6, 7 and 8 can be analyzed in relation to the time between strokes and sample collection, or alternatively, in relation to the type of stroke: ischemic, hemorrhagic or transient ischemic attack (TIA). These separate analyzes are shown in Figure 25a and Figure 25b and demonstrate the usefulness of CSF markers of deceased in the diagnosis of stroke. This is particularly relevant for clinical practice, since it is essential to diagnose

40 the stroke within three hours of the event to allow the administration of anticoagulant medications, such as TPA. It is also essential that tests can differentiate a hemorrhagic stroke from ischemic attack, since TPA is only suitable for the treatment of ischemia and can have catastrophic effects in patients with hemorrhagic stroke.

45 EXAMPLE 10

While each of the CSF markers of deceased has a good individual performance for the diagnosis of stroke, it is likely that a commercial product requires the measurement of the levels of several proteins. This "panel" approach can be achieved in two ways. In the simplest approach, the antibodies for each

50 separate markers are grouped and used to coat microtiter wells. The signal strength will be the sum of that for each independent marker, although in this case, it will be impossible to determine the individual levels of each of the markers. This can create challenges when establishing significant cut-off values, however, this presents the most respectful commercial product with the user.

55 Figure 26 summarizes the markers used in this Example. Experimental results are shown in Figure 27, in which antibodies against CSF proteins of deceased UFD1, RNA-BP, NDKA and H-FABP were used at the same concentrations as in Example 4. However, these solutions of antibodies were mixed in equal volumes, reducing the concentration of each antibody species to a quarter of the original level in the individual analyte examples described above. The protocol used is as indicated by

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Claims (1)

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