Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/28—Neurological disorders
  • G01N2800/2814—Dementia; Cognitive disorders
  • G01N2800/2821—Alzheimer

Definitions

• AD Alzheimer's disease
• ⁇ APP a neurodegenerative disease of the central nervous system
• ⁇ APP a neurodegenerative disease of the central nervous system
• a first is Familial Alzheimer' s Disease (FAD) which is genetically linked, generally exhibits an early onset and may be caused by a faulty gene for the Alzheimer's precursor protein, ⁇ APP, on chromosome 21 leading to an altered ⁇ APP sequence.
• FAD Familial Alzheimer' s Disease
• AD Alzheimer's disease
• ⁇ APP amyloid precursor protein
• Platelets are one of several cellular components of blood and when appropriately stimulated, exhibit a response that includes, inter alia, a change in the membrane potential of the platelet, changes in the intra-cellular Ca + + and a secretion or release of granules and the granules' contents.
• platelet activation in response to physiological stimuli such as ⁇ -thrombin (less than 4.5nM) has been extensively studied.
• Cf. for example, Davies et al, (1987) Analyt.
• a principal object of the present invention is to provide an assay for Alzheimer' s disease in live patients.
• the invention herein described and claimed generally then is a method of assaying for non-familial Alzheimer' s disease in a live patient, comprising the steps of collecting platelets from the blood of said patient and stimulating those platelets with a physiological agonist, typically ⁇ -thrombin, in order to invoke a characteristic acidification response in the platelet cytoplasm.
• a physiological agonist typically ⁇ -thrombin
• Other known agonists include, but are not limited to, collagen, adenosine disphosphate, epinephrin, ristocetin and the like.
• the extent of that response immediately following stimulation is determined, preferably with a pH- sensitive probe such as fluorescent material which can be emplaced within the platelet cytoplasm.
• That measured extent of response is then compared with the corresponding stimulated acidification response in platelets from normal individuals, i.e- those apparently not suffering from non-familial Alzheimer's disease.
• Stimulation of human platelets by thrombin is known to be accompanied by, inter alia, a rapid thrombin-dose-dependent cytoplasmic acidification or change in the pH;., within a few seconds (e.g. 3 or 4 sec.), followed by a cytoplasmic alkalinization that maximizes quickly.
• the extent of cytoplasmic acidification can then be determined using any of a number of pH sensitive techniques such as with fluorescent probes including 6-carboxyfluorescein (hereinafter 6-CF), 2 * ,7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (hereinafter BCECF) and the like.
• 6-CF 6-carboxyfluorescein
• BCECF 2 * ,7'-bis(carboxyethyl)-5(6)-carboxyfluorescein
• the esters of these fluorescein derivatives are non-fluorescent and, in this form, pass freely across the platelet plasma membrane. Inside the cell, the ester group is believed to be cleaved by nonspecific cytoplasmic esterases, generating an in situ pH-sensitive fluorescein probe.
• BCECF is preferred as a probe over 6-CF inasmuch as the former has a higher pK (6.9) and is pH sensitive to 7.8 or 7.9.
• Thrombin-induced activation of platelets was measured by signal transduction and degranulation for younger individuals (hereinafter Control) and age-matched older normal (AM) and Alzheimer's diseased patients (AD) as identified by the criteria developed by the Alzheimer's Disease and Related Dementia Association in conjunction with the National Institute of Aging (McKhann et al (1984) Neural. 34, 939). Neutrophils isolated from the same blood samples were used as controls.
• the neutrophils from all three groups exhibited identical responses (depolarization and oxidative burst) to the chemotactic peptide formyl methionine leucine phenylalanine, indicating that the platelet difference in AD patients are not attributable to any artifact in the patient's blood per se. Additionally, the platelet resting parameters, pH.-, ⁇ and [Ca ++ ] rejting , in the three groups were not significantly different. However, the maximum change in cytoplasmic thrombin-induced acidification, usually reached within the first few seconds (e.g. ca.
• Sepharose 2B a sugar polymer used in column chromatography and serving as a selective filter that permits the larger particles to pass through while retaining the smaller particles
• the fluorescent probes such as 2',7'-bis (carboxyethyl)- 5(6)- carboxyfluorescein-acetoxymethyl ester (hereinafter BCECF-AM) and 5(6)-carboxyfluorescein diacetate (6-CF), as well as the ionophore nigericin, were obtained from Molecular Probes, Inc. of Eugene, Oregon.
• HEPES 4-(2 hydroxyethyl)-l-piperazine ethane-sulfonic acid
• K + -Hepes buffer 0.140 M KC1, 0.0038 M HEPES, 0.0055 M D-glucose, 0.0033 M monobasic sodium phosphate, 0.001 M MgCl 2 -6H 2 0, 0.15U/liter apyrase, pH 6.6 - 7.6.
• PLATELETS Fresh whole blood from volunteers was anticoagulated with 0.38% sodium citrate. Platelet-enriched plasma was obtained by centrifugation, and the platelets separated by passage over a Sepharose 2B column equilibrated with a Hepes buffer (pH 7.4) as described in Home et al (1981) Eur. J. Biochem. 120, 295-302. EXAMPLE I.
• the resuspended pellets were then diluted to a final concentration of about 30xl0 6 platelets/ml with either Hepes buffer, pH 7.4, for thrombin response measurements, or K + -Hepes buffer at varying pHs, immediately before use, for calibration curves.
• Hepes buffer pH 7.4, for thrombin response measurements, or K + -Hepes buffer at varying pHs, immediately before use, for calibration curves.
• ⁇ -thrombin 0.0025-0.05 U/ml, 0.45-9 nM
• Intracellular pH was determined from a calibration curve prepared using the same ratio procedure as described by Thomas et al, (1979) Biochemistry 18, 2210-2218, correlating the difference at set pH values (pH 6.6 to 7.6) with ratios obtained when cells, incubated in K + - Hepes buffer are measured before and after the addition of nigericin (Davies et al, (1987) Analyt. Biochem. 167, 118-123).
• a relative rate of cytoplasmic pH change can also be determined by measuring the initial slope of the thrombin induced fluorescence change (cm/min), divided by the initial F 450 (i.e. initial pH-insensitive ) value for the probe (Davies et al (1990) J. Biol. Chem. 265, 11522).
• the rate and extent of cytoplasmic acidification for the three groups for different doses of thrombin is given in the following Table in which the rate of cytoplasmic acidification was ⁇ pH/ ⁇ t in reciprocal seconds, and the extent of acidification was measured as ⁇ pH.
• Example II Intracellular pH was determined from a calibration curve prepared as described in Example III, and the data substantiated the data given in Table I for controls. EXAMPLE III. The method described in Example I was repeated, however, 2 ⁇ M nigericin, instead of thrombin was added to the platelets in order to prepare a pH calibration curve, and platelets were diluted in K + -Hepes at varying pH (6.6 to 7.6) instead of normal Hepes. The change in ratio was calculated as follows:

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• 1993
  • 1993-06-18 EP EP93916630A patent/EP0649474A4/de not_active Withdrawn
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