WO2005043136A1 - Methode de diagnostic precoce de maladies prioniques par spectroscopie infrarouge - Google Patents

Methode de diagnostic precoce de maladies prioniques par spectroscopie infrarouge Download PDF

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WO2005043136A1
WO2005043136A1 PCT/ES2004/070091 ES2004070091W WO2005043136A1 WO 2005043136 A1 WO2005043136 A1 WO 2005043136A1 ES 2004070091 W ES2004070091 W ES 2004070091W WO 2005043136 A1 WO2005043136 A1 WO 2005043136A1
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fraction
proteins
blood
prp
identification
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PCT/ES2004/070091
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English (en)
Spanish (es)
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Pedro Carmona Hernandez
Jaime Monreal Llop
Eva MONLEÓN MOSCARDÓ
Marta MONZÓN GARCÉS
Juan José BADIOLA DÍEZ
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Consejo Superior De Investigaciones Científicas
Universidad De Zaragoza
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    • 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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/359—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using near infrared light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • 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
    • G01N2800/2828—Prion diseases

Definitions

  • the invention concerns a first priority sector corresponding to the area of food security and livestock, with subsequent application to a second sector of health. More specifically, the invention relates to an early diagnosis method for prion diseases based on infrared spectroscopy.
  • TSEs Transmissible Spongiform Encephalopathies
  • c) Present, mainly in the central nervous system (CNS), the accumulation of an abnormal isoform (PrP Sc ) of a membrane glycoprotein (PrP c ) encoded by the host itself that differs from the normal one in its conformation (specifically, in a greater percentage than it presents of structure of folded sheet ⁇ ).
  • d) Produce a characteristic picture of lesions, consisting of a spongiform change that affects, mainly the brainstem, both the neuropil of the gray matter and the pericarion of the neurons.
  • the scrapie or scrapie first described in 1732 in the United Kingdom and later diagnosed in sheep and goats in a large number of countries, is the most widely studied animal TSE, and is considered a prototype of this group of diseases.
  • Bovine Spongiform Encephalopathy (BSE; United Kingdom, 1986) has acquired greater relevance due to its implications in Public Health, after the appearance of the new Creuzfeldt-Ja-kob variant (v-CJD; United Kingdom, 1996) in humans associated with consumption of meat products contaminated with the BSE agent.
  • Creutzfeldt-Jakob disease sporadic, iatrogenic and familial; variant of Creutzfeldt-Jakob disease (v-CJD); Kuru; Gerstmann syndrome-
  • GSS Straüssler-Scheinker
  • FMI Lethal Insomnia
  • FFI Lethal Insomnia
  • ⁇ -fibrilosis Gajdusek, Nutrition, Health and Peace Linus Pauling Inst, Palo Alto, 21-55, 1987
  • ⁇ configuration characterized by the accumulation of neurotoxic proteins of ⁇ configuration, among which Alzheimer's disease is the most characterized and spread.
  • TSEs human and animal
  • OIE International Organization of Epizootics; Regulation 1248/2001
  • histopathology and immunochemical techniques --- tmunotastochemistry and western blotting
  • Scrapie associated flbril SAF
  • Infrared spectroscopy in its modern instrumentation, is a powerful and rapid technique for the analysis of secondary structure of proteins, since it allows distinguishing and quantifying structures, ⁇ and disordered. Since infectious prion proteins (PrP Sc ) have a high content of ⁇ structure with respect to the normal prion isoform, infrared spectroscopy, used according to the procedures of this invention, allows to identify PrP Sc proteins in blood samples from sheep animals affected by scrapie, even in preclinical phase. There is no background in the literature on infrared spectroscopy work focused on the analysis of prion proteins in blood.
  • infrared microspectroscopy has been used to differentiate brain tissue from healthy and affected scrapie rodents (Kenipp et al., Biochim. Biophys. Acta, 1501, 189-199 (2000); Naumann et al., Patent WO 00 / 72007 A2, Nov 30, 2000; Kneipp et al., J. Neuroscience, 22, 2989-2997 (2002)).
  • DESCRIPTION OF THE INVENTION Brief Description of the Invention The present invention relates to a method, by infrared spectroscopy, for the detection of infectious prion proteins (PrP Sc ) in biological samples from living animals affected by a naturally acquired TSE.
  • infective prion proteins in a sample can be determined by calculating the percentages of ⁇ structure, which undergoes a significant increase in these infective proteins with respect to the controls corresponding to healthy animals.
  • This quantitative identification method is also part of the present invention.
  • the analytical results obtained according to this invention have been 100% agreed on about 100 investigated animals and whose results have been validated with the post mortem diagnostic tests admitted by the OIE of CNS samples carried out on the corresponding animals from which samples were previously obtained. blood.
  • the biological sample for performing this procedure can come from a biological fluid, such as blood, serum, cerebrospinal and lymphatic fluid, and urine, or from tissue, either as such, or homogenized or solubilized by standard procedures.
  • a biological fluid such as blood, serum, cerebrospinal and lymphatic fluid, and urine
  • tissue either as such, or homogenized or solubilized by standard procedures.
  • the living beings object of analysis are, among others, sheep, cows, goats, mouflon, mink, deer, cats, rodents, and humans.
  • This procedure has application for the diagnosis of preclinically or clinically infected animals with TSEs and humans affected by other ⁇ -fibrillosis, and the study of the evolution of the infection, among others, of scrapie in sheep and goats, mouflon, BSE, the chronic cacketizing deer disease, all variants of CJD disease, Gerstmann-Stráussler-Scheinker disease, and fatal familial insomnia, and their use for such purposes has been claimed in the present invention.
  • the present invention is based on the fact that the authors have observed that infectious prion proteins (PrP Sc ) can be identified, qualitatively and quantitatively, specifically from biological samples from infected animals. Said presence can be verified in a differentiated way in the same animal that suffers a prion infection over time, after a specific process of obtaining said infective prion proteins in a cellular fraction of the blood and their subsequent detection by spectroscopy. infrared.
  • the present invention provides a new method of detection of infectious marker prion proteins, PrP Sc , on which the diagnosis of TSEs in animals is based, and which comprises obtaining a sample enriched in PrP Sc , from animal biological samples and identification, by spectroscopy infrared, of the PrP So marker proteins in said sample.
  • the procedure comprises the following steps: 1) obtaining a fraction from the blood and its blood cells subjected to lysis with water and that after centrifugation the resulting pellet is enriched in PrP Sc (procedures a), b) and c)); 2) H / D (hydrogen / deuterium) isotopic exchange in each of the cell fractions obtained; 3) the identification, by infrared spectroscopy, of the PrP Sc marker proteins in said lysate fraction.
  • the term "animals” refers to those species that can be infected by PrP Sc proteins, among others, sheep, cows, goats, mouflon, mink, deer, rodents, cats and humans.
  • biological sample in the present invention refers not only to biological fluids from animals, for example: blood, serum, plasma, cerebrospinal and lymphatic fluid, and urine, but also biological samples from tissues, which have been homogenized by mechanical procedures, sonication or any other procedure known in the state of these techniques, and solubilized.
  • the term “cellular element lysate fraction” refers to the fraction of the biological sample from which the infectious prion proteins are divided and concentrated.
  • a particular objective of the present invention is a method of detecting infectious prion proteins, by means of which, obtaining a lysate fraction of cellular elements from biological samples (blood) of animals, enriched in PrP Sc , (procedure a) of the present invention (see Examples 1 and 2), comprises the following steps: - extraction of sheep blood, 8-10 ml, with 1.4 mM EDTA, in sterile tubes. refrigerated centrifugation and at 4,300 g (6,000 rpm) for 30 minutes, of the 8-10 ml of blood placed in tubes of approximately 40 ml.
  • Procedure b) Another particular objective of the present invention is a much faster preparation method than the method a) of detecting infective prion proteins.
  • obtaining a lysate fraction of cellular elements from biological samples (blood) of animals, enriched with PrP So proteins, comprises the following steps: - Starting from approximately 40 ml, containing approximately 3/4 of its Milli-Q water capacity.
  • Procedure c 10 ml of blood with recently extracted 1.4 mM EDTA are placed in a polystyrene tube with a conical bottom and a 15 ml capacity sterile screw cap, and centrifuged in an oscillating rotor at 1,250 g for 15 minutes, obtaining at the interface white cells (fraction A, buffy coat). - The interface is collected (fraction A, approximately 1 ml) and added to 6 ml of TBS 0.05M Tris / HCl, 0.15M NaCl, pH 7.6. In a suitable tube the gradient is formed, consisting of 3 ml of Ficoll-Paque TM PLUS solution (Sweden), on which the 7 ml of fraction A are placed suspended on TBS.
  • Ficoll-Paque TM PLUS solution Surden
  • fraction C interface
  • fraction B pellet
  • erythrocytes and polymorphonuclear cells neutralils and to a lesser extent, eosinophils and basophils
  • Figure 9 (b) eosinophils and basophils
  • Fractions A and B are osmotic with 25 ml of Milli-Q water in a tube (Falcon ) of 50 ml conical bottom, and then 25 ml of a saline solution (1.8% NaCl) are added; subsequent washes (x3) with 25 ml of TBS by centrifugation at 500 g for 10 min; finally the pellet is resuspended in TBS in an ependorf tube for possible analysis or freezing - About 1 ml of cell suspension is collected from the interface (fraction C), which is collected in a 1 ml ependorf tube It is centrifuged at maximum speed for 10 minutes in a microcentrifuge at 4 ° C , the supernatant is discarded and the resulting pellet is resuspended in Milli-Q water for the corresponding osmotic shock.
  • the mini-dialyzer which contains the dialysate membrane at the base thereof, is allowed to float in a small 10 ml beaker in which several ml of heavy water are deposited.
  • the vessel is tightly covered with parafilm paper, and dialysis is carried out by shaking for 30 minutes so that the isotopic exchange of the water protons and the amide protons of the protein polypeptide skeleton is achieved.
  • the amide band I of the proteins is free from the influence of the molecular species H 2 O, whose angular deformation band (scissoring) moves by deuteration towards 1200 cm "1.
  • Proteins with ⁇ structure such as infective prions, generate infrared absorption bands close to 1637 cm “1 , which has been demonstrated by means of spectra on patterns of these proteins (Caughey et al., Biochemistry, 48, 32230-32235 (1998 ); Pan et al., Proc. Nati. Acad.
  • FIGURES Figure 1. Infrared spectra of a pellet, preferably consisting of used white cells and their membranes from the blood of a healthy sheep (control), validated by negative biopsy and autopsy, and obtained by procedure a. Original spectrum (upper), and second derivative spectrum (lower).
  • Giemsa staining of blood fraction C obtained by processing c) from an sheep affected by scrapie disease consisting mainly of mononuclear cells (lymphocytes preferably and to a lesser extent monocytes) (x 400).
  • Figure 9 (b). Giemsa staining of blood fraction B obtained by processing c) from an sheep affected by scrapie disease, composed mainly of polymorphonuclear cells (preferably neutrophils, and to a lesser extent eosinophils and basophils) (x 400).
  • Example 1 Infrared spectroscopy of pellets. from Used cell phones. obtained from healthy sheep blood (control), from procedure a). Following this procedure, approximately 5-10 ⁇ l of deuterated pellet were introduced between two CaF crystals with a 25 ⁇ Teflon separator. The spectrum obtained, measured on a FTIR Perkin-Elmer spectrometer model 1725X, was the result of averaging 64 scans at a resolution of 2 cm "1 and subtracting the spectrum of heavy water under the same experimental conditions.
  • the first 8 animals correspond to negative sheep controls, and those numbered from 9 to 23 correspond to sheep affected by scrapie.
  • Animal No. 24 is a negative control goat (Saanen breed) and Nos. 25 and 26 (Crosses and Alpine breeds, respectively) correspond to scrapie-infected goats.
  • the vast majority of sheep analyzed are female from Rasa Aragonesa.
  • the genotype is in most sheep is ARQ / ARQ, only two ARQ / ARH and one ARQ / VRQ. * These numbers correspond to the same animal as that of the immediately previous number, but blood was drawn 1-2 months later.
  • CC clinical picture at the time of blood collection.
  • NT not tested. Necropsies not performed are because the respective animals have not been slaughtered.
  • SNC / SLR Central Nervous System / Lymphoreticular System.
  • Example 2 Infrared spectroscopy of pellets from Cellular uses, obtained from the blood of infected sheep, from procedure a).
  • Figure 2 includes the infrared spectrum of the pellet from the cell lysate fraction obtained by procedure a), corresponding to an animal affected by scrapie. The spectroscopic measurements were carried out under the same experimental conditions as in Example 1.
  • a band towards 1636 cm "1 is observed which is much more intense than that corresponding to the spectrum of Figure 1 ( negative control), and that can be attributed to ⁇ structures of prion proteins.
  • the infrared spectra corresponding to patterns of these prion proteins (Caughey et al., J.
  • samples 10 and 13 of Table 1 from the same animal as the previous immediate samples (9 and 12 respectively), but extracted 1-2 months later, have a higher ⁇ -structure content. This allows to follow, by infrared spectroscopy, the evolution in aggravation of the animal disease. It should also be noted that there are cases, such as those corresponding to samples numbers 12 and 13 of Table 1, in which the biopsies were initially negative in the first blood draw and, however, the infrared analysis resulted in percentages of structure ⁇ typical of infected animals, which was subsequently confirmed by post mortem autopsy. This case is indicative that this spectroscopic technique is even capable of giving a preclinical diagnosis of scrapie. which, however, the third eyelid biopsy has not been able to detect.
  • infrared spectroscopy is capable of detecting infectivity, based on the percentages of ⁇ structure found. This infectivity, as noted in this table, is confirmed with the corresponding positive biopsies and / or necropsies. Using this spectroscopic methodology, given the duration of the spectra measurement, approximately one hundred blood samples per day can be analyzed if they are previously prepared (fractionated and isotopically exchanged). Example 3. Infrared spectroscopy of sample pellets from
  • Figure 3 presents an infrared spectrum of a pellet of healthy sheep cell lysate (negative control) obtained by procedure b).
  • the most prominent bands are those located around 1660, 1651 and 1644 cm “1 , attributable to bearing structures, ⁇ - helices and disordered ones
  • the percentages of ⁇ structure of this negative control and five more correspond to values that are, similarly to Table 1, below 10%. Described in the following example, these percentages are lower than those corresponding to blood samples from infected animals.
  • Example 4 Infrared spectroscopy of pellet samples from Cellular uses, obtained from the blood of infected sheep, from procedure b
  • Figure 4 shows the infrared spectrum of a deuterated pellet, measured under the same conditions as in the previous examples, and corresponding to an infected sheep blood cell lysate obtained by procedure b).
  • Example 5 Infrared spectroscopy of white blood cells (Fraction A), obtained by procedure c).
  • Figures 5 and 6 include the spectra of Fraction A (buffy coat), preferably composed of white blood cells, corresponding respectively to a healthy (negative control) and infected animal.
  • the most significant qualitative differences between these figures include the intensities of the bands in the spectra of the second derivatives, particularly the bands corresponding to the ⁇ structures that appear around 1638 and 1631 cm "1. In the case of the infected sample ( Fig. 6), these bands appear much more intense than in the negative control spectrum (Fig. 5) On the other hand, it is also interesting the fact that in Fig.
  • Example 6 Infrared spectroscopy of white blood cells (Fraction C). obtained by procedure c).
  • Figures 7 and 8 show respectively the white cell infrared spectra (mostly Unfocytes and to a lesser extent, monocytes) obtained in Fraction C of healthy sheep and affected by scrapie.
  • the bands generated by the prion ⁇ structures around 1637 and 1631 cm “1 which in the case of the control spectrum appear very weak and yet are much more intense in the spectrum of the infected animal.
  • the existence of several isoforms of ⁇ -prion structure corresponding to the mentioned bands is also observed (1637, 1631 and 1626 cm "1 ).
  • the frequency of the band towards 1618 cm-1 may correspond to prion proteins that are aggregated in the isolated cellular elements.
  • infective prion proteins are preferably located in the blood cell fraction C, consisting mainly of lymphocytes.

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Abstract

La présente invention concerne une méthode de détection in vivo de protéines prioniques infectieuses dans le sang d'animaux souffrant d'encéphalopathie spongiforme transmissible, par spectroscopie infrarouge.
PCT/ES2004/070091 2003-10-31 2004-10-27 Methode de diagnostic precoce de maladies prioniques par spectroscopie infrarouge WO2005043136A1 (fr)

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ES200302561A ES2246113B1 (es) 2003-10-31 2003-10-31 Procedimiento de deteccion ante-mortem de proteinas prionicas por espectroscopia infrarroja y su uso en el diagnostico de encefalopatias espongiformes transmisibles.
ESP200302561 2003-10-31

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9891108B2 (en) 2012-10-03 2018-02-13 The Research Foundation For The State University Of New York Spectroscopic method for Alzheimer's disease diagnosis

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2396106B1 (es) * 2011-08-08 2014-06-06 Consejo Superior De Investigaciones Científicas (Csic) Análisis infrarrojo de fracciones de sangre periférica obtenida para indicar desarrollo cognitivo asociado a la enfermedad de alzheimer.

Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2000065357A1 (fr) * 1999-04-21 2000-11-02 Boehringer Ingelheim Vetmedica Gmbh Methode de diagnostic des encephalopathies spongiformes transmissibles
WO2000072007A2 (fr) * 1999-05-20 2000-11-30 Robert-Koch-Institut Procede pour diagnostiquer par spectroscopie infrarouge des modifications de tissu induites par une encephalopathie spongiforme transmissible (tse)
WO2002066963A2 (fr) * 2001-02-22 2002-08-29 Bundersrepublik Deutschland Vertreten Durch Das Bundesministerium Für Gesundheit, Dieses Vertreten Durch Das Rober-Koch-Institut Vertreten Durch Seinen Leiter Procede de detection de modifications induites par l'encephalopathie spongiforme chez l'homme et l'animal

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000065357A1 (fr) * 1999-04-21 2000-11-02 Boehringer Ingelheim Vetmedica Gmbh Methode de diagnostic des encephalopathies spongiformes transmissibles
WO2000072007A2 (fr) * 1999-05-20 2000-11-30 Robert-Koch-Institut Procede pour diagnostiquer par spectroscopie infrarouge des modifications de tissu induites par une encephalopathie spongiforme transmissible (tse)
WO2002066963A2 (fr) * 2001-02-22 2002-08-29 Bundersrepublik Deutschland Vertreten Durch Das Bundesministerium Für Gesundheit, Dieses Vertreten Durch Das Rober-Koch-Institut Vertreten Durch Seinen Leiter Procede de detection de modifications induites par l'encephalopathie spongiforme chez l'homme et l'animal

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Title
CAUGHEY B. ET AL.: "Strain-dependent differences in B-sheet conformations of abnormal prior protein", J. BIOL. CHEM., vol. 273, no. 48, November 1999 (1999-11-01), pages 3230 - 3235 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9891108B2 (en) 2012-10-03 2018-02-13 The Research Foundation For The State University Of New York Spectroscopic method for Alzheimer's disease diagnosis

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