EP1381868A2 - Method for testing samples containing prion protein for the possible presence of the prp?sc form - Google Patents

Method for testing samples containing prion protein for the possible presence of the prp?sc form

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Publication number
EP1381868A2
EP1381868A2 EP02737971A EP02737971A EP1381868A2 EP 1381868 A2 EP1381868 A2 EP 1381868A2 EP 02737971 A EP02737971 A EP 02737971A EP 02737971 A EP02737971 A EP 02737971A EP 1381868 A2 EP1381868 A2 EP 1381868A2
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Prior art keywords
prp
sample
prion protein
protease
molecules
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EP02737971A
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German (de)
French (fr)
Inventor
Karin Biffiger
Markus Moser
Bruno Oesch
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Prionics AG
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Prionics AG
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Publication of EP1381868A2 publication Critical patent/EP1381868A2/en
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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
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases

Definitions

  • the invention relates to a method according to the preamble of claim 1.
  • Generic methods are currently especially in mammalian screening studies, e.g. Slaughter cattle, applied to communicable, degenerative neurological diseases.
  • Such diseases which are summarized under the term spongiform encephalopathies or prion diseases, occur as BSE in cattle and e.g. as scrapies in sheep, or as Kuru or Creuzfeldt-Jakob disease in humans.
  • prion diseases are communicable, although the infectiousness has not yet been fully clarified.
  • the only molecule associated with the infectious agent has so far been found to be a disease-specific prion protein (PrP c ) which is an abnormal isoform of a normal mammalian protein (PrP c ) of unknown function.
  • PrP c disease-specific prion protein
  • Both isoforms PrP Sc and PrP c are the same in terms of molecular weight and amino acid sequence. They differ in their spatial folding.
  • PrP Sc proteins are able to convert normal PrP c proteins into disease-specific folding, which would explain the infectivity of PrP Sc proteins.
  • PrP Sc the central disease molecule
  • samples from infected sources contain not only PrP Sc but also prion protein in the PrP c form. During the process, a differentiation must therefore be made between the PrP c and the possibly existing PrP Sc form.
  • PrP c form is completely protease digestible, while in the PrP c form only one C-terminal region is protease sensitive and one region of the prion protein protease, designated as PrP 27-30, is resistant is.
  • the sample to be examined is therefore first digested with a protease in a first step (step a), it being assumed that after digestion, no protease-sensitive areas of the prion protein are present in the sample and only in the case of infectious sample material the protease resistant region PrP 27-30 of the PrP Sc form remains. Accordingly, after the digestion, in a further step (step b) it is only checked whether the area PrP 27-30 can be detected in the sample or not. For example, specific antibodies binding in the range PrP 27-30 are used for detection. Any antibody-PrP 27-30 complexes formed are then detected using conventional methods, for example Elisa tests (Moynagh and Schimmel; Nature 1999 Jul 8; 400 (6470): 105). If the proof is positive, ie if antibody-PrP 27-30 complexes can be detected, it is assumed that the sample contains PrP Sc and that the organism from which the sample comes was infected.
  • the object of the invention is to develop generic methods so that a more reliable statement is possible.
  • the sample in step b is not only checked to determine whether it contains the PrP 27-30 region, but also to check whether protease is still present in the sample. sensitive areas of the prion protein are contained.
  • the method according to the invention thus allows a statement about the possible presence or absence of PrP 27-30 in the digested sample and a statement as to whether the digestion was complete or not.
  • PrP 27-30 in a digested sample is only considered as an indication of PrP Sc if none of the digested sample is present at the same time Protease sensitive areas of the prion protein are more detectable. If such protease sensitive areas can still be detected in the sample even after digestion, the possible detection of PrP 27-30 is not a clear indication of PrP Sc but could also mean that a corresponding area of the PrP c form was not completely digested. In this case the sample would have to be examined again with, for example, higher protease concentrations or longer digestion times.
  • false positive examination results can be excluded in a particularly safe and simple manner.
  • the significance of a test essentially depends on the fact that false positive results are minimized.
  • PrP 27-30 or protease-sensitive regions of the prion protein are detected by means of molecules which bind specifically to the prion protein in the respective regions and which are described below as molecule A (specific for a protease-sensitive) Area) and molecule B (specifically for the area PrP 27-30).
  • a common method provides that in step a the digestion of the sample takes place and in step b the molecules A and B of the digested sample are added and it is checked whether there are complexes from the prion protein with the molecule in the sample A and / or B have formed. The evaluation is then carried out depending on whether and which complexes have formed.
  • the sample contains PrP Sc . Are there additional complexes that contain the molecule A there is a risk of a false positive result. If no complexes or only complexes with molecule A are found, the sample is negative.
  • Molecules A and B can in particular be antibodies (hereinafter referred to as antibodies A and B) which specifically recognize the corresponding regions of the prion protein.
  • antibodies A and B antibodies which specifically recognize the corresponding regions of the prion protein.
  • other specifically binding molecules e.g. RNA molecules are used.
  • Antibodies which recognize the protease-sensitive N-terminal region of the PrP are, for example, from "Brain Research, 545, (1991) 319-321 (antiserum anti-PrP-N),” Brain Pathol. 2002; 12: 1-11 (Antibodies FH11, BG4) "," Proc. Natl. Acad. Be. Vol. 95 pp. 8812-8815, July 1998 (Antikö ⁇ er 5B2) M or "Biochemical and Biophysical Research Communications 273, 136-139 (2000) (Antikö ⁇ er 8B4)".
  • the publications mentioned describe the properties of the antibodies and their production.
  • the complexes formed can be verified as standard. It is usually provided that one of the two components of the complex formed is carrier-bound.
  • the sample material is immobilized after digestion, for example on a microtiter plate or on beads, and a detection with labeled molecules A and B, in particular antibodies A and B, is carried out.
  • the preferably used antibodies can be incubated in one batch simultaneously or in succession with the sample material. It is equally possible to carry out two parallel runs, in each of which one of the two antibodies A or B is added.
  • a sandwich immunoassay is preferably carried out for detection.
  • two antibodies per analyte are used in such sandwich immunoassays, which are bound to two different epitopes of the analyte.
  • One of the antibodies is usually immobilized and serves to couple the analyte to a solid phase, while the other serves as a detection antibody and is provided with a label.
  • a further antibody C is also provided in this context, which recognizes PrP 27-30, although the epitope recognized by this antibody C is different than that recognized by Antikö ⁇ er B.
  • Another possibility is to immobilize the antibodies A and B on a support and then, after incubation of the support with the sample material, to add labeled antibody C for detection.
  • the sample material obtained after digestion is incubated in one batch first with immobilized antibodies A and then with immobilized antibodies B. Labeled antibody C is added for detection as described above.
  • the staggering over time makes it possible in a simple manner that any protease-sensitive areas of the PrP that are present can initially bind to the specific antibodies A, without the binding kinetics being caused by a simultaneous attack by the antibodies serving as molecule B on the protease-resistant Area is disturbed.
  • any complexes formed are detected using labeled molecules, in particular labeled antibodies. If a label can be detected or recognized on a support, this is a sign that the relevant antibody provided with the detected label has been bound, which, depending on the test setup, can be an indication of the presence of a certain complex.
  • the molecules A and B or the antibody C can be e.g. the same or different fluorescent labels or with enzymes (Elisa) or with other suitable labels. Basically, all markings are suitable that can be directly or indirectly detected or measured.
  • the person skilled in the art is aware of the different possibilities for labeling molecules, in particular antibodies, in a suitable manner for the above processes and for detecting them in the course of the processes. It should therefore not be dealt with in more detail at this point

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The invention relates to a method for testing samples containing prion protein for the possible presence of the PrPSc form, according to which: (Step a) the sample is mixed with protease in order to digest protease-sensitive proteins or protein regions; (Step b) after digestion, it is tested whether the sample contains the region PrP 27-30 of the prion protein, which is resistant in the PrPSc form of the prion protein protease, and the presence of PrPSc in the sample is established based on the positive detection of PrP 27-30. The inventive method is characterized in that, during Step b, the sample is additionally tested in order to determine whether a complete digestion of the protease-sensitive region of the prion protein has occurred.

Description

Verfahren zur Untersuchung von Prion-Protein enthaltenden Proben auf das eventuelle Vorliegen der PrPSc-FormMethod for examining samples containing prion protein for the possible presence of the PrP Sc form
Die Erfindung bezieht sich auf ein Verfahren nach dem Oberbegriff des Anspruches 1.The invention relates to a method according to the preamble of claim 1.
Gattungsgemäße Verfahren werden z.Zt. insbesondere bei Screening- Untersuchungen von Säugetieren, z.B. Schlachtvieh, auf übertragbare, degenerative neurologische Erkrankungen angewandt. Solche Erkrankungen, die unter dem Begriff spongiforme Enzephalopatien oder auch Prionerkrankungen zusammengefaßt werden, treten als BSE bei Rindern und z.B. als Scrapies bei Schafen, oder als Kuru oder Creuzfeldt- Jakob-Krankheit bei Menschen auf.Generic methods are currently especially in mammalian screening studies, e.g. Slaughter cattle, applied to communicable, degenerative neurological diseases. Such diseases, which are summarized under the term spongiform encephalopathies or prion diseases, occur as BSE in cattle and e.g. as scrapies in sheep, or as Kuru or Creuzfeldt-Jakob disease in humans.
Wie oben erwähnt sind Prionerkrankungen übertragbar, wobei die Infektiösität noch nicht vollständig geklärt ist. Als einziges mit dem infektiösen Agens assoziiertes Molekül wurde bislang ein krankheitsspezifisches Prion-Protein (PrP c) gefunden, das eine abnorme Isoform eines normalen Säugetierproteins (PrPc) unbekannter Funktion ist. Beide Isoformen PrPSc und PrPc stimmen bezüglich Molekulargewicht und Aminosäuresequenz überein. Sie unterscheiden sich in ihrer räumlichen Faltung.As mentioned above, prion diseases are communicable, although the infectiousness has not yet been fully clarified. The only molecule associated with the infectious agent has so far been found to be a disease-specific prion protein (PrP c ) which is an abnormal isoform of a normal mammalian protein (PrP c ) of unknown function. Both isoforms PrP Sc and PrP c are the same in terms of molecular weight and amino acid sequence. They differ in their spatial folding.
Viele Indizien, insbesondere die Abwesenheit von anderen Molekülen außer PrPSc im Prion und vor allem die Abwesenheit von Nukleinsäuren, deuten daraufhin, daß PrP c wahrscheinlich die zentrale Rolle bei der Auslösung der oben genannten Krankheiten zukommt. Es wird angenommen, daß PrPSc-Proteine in der Lage sind, normale PrPc-Proteine in die krankheitsspezifische Faltung zu konvertieren, was die Infektiösität von PrPSc-Proteinen erklären würde.Many indications, in particular the absence of molecules other than PrP Sc in the prion and especially the absence of nucleic acids, indicate that PrP c probably plays the central role in triggering the above-mentioned diseases. It is believed that PrP Sc proteins in are able to convert normal PrP c proteins into disease-specific folding, which would explain the infectivity of PrP Sc proteins.
Gattungsgemäße Tests gehen daher von PrPSc als zentralem Krankheitsmolekül aus und überprüfen, ob das z.B. in einer Hirnprobe eines Säugetiers enthaltene Prion-Protein auch in der PrPSc-Form vorliegt. Falls ja, wird davon ausgegangen, daß das Säugetier, dem die Probe entnommen wurde, infiziert war.Generic tests are therefore based on PrP Sc as the central disease molecule and check whether the prion protein contained in a brain sample of a mammal, for example, is also present in the PrP Sc form. If so, it is assumed that the mammal from which the sample was taken was infected.
Dabei enthalten, wie erwähnt, Proben aus infizierten Quellen nicht ausschließlich PrPSc sondern daneben auch Prion-Protein in der PrPc-Form .Während des Verfahrens muß also eine Differenzierung zwischen der PrPc und der eventuell vorliegenden PrPSc-Form erfolgen.As mentioned, samples from infected sources contain not only PrP Sc but also prion protein in the PrP c form. During the process, a differentiation must therefore be made between the PrP c and the possibly existing PrP Sc form.
In diesem Zusammenhang macht man sich zunutze, daß die PrPc-Form vollständig Protease-verdaubar ist, während bei der PrP c-Form nur ein C-terminaler Bereich Protease sensitiv und ein als PrP 27-30 bezeichneter Bereich des Prion- Proteins Protease resistent ist.In this context, use is made of the fact that the PrP c form is completely protease digestible, while in the PrP c form only one C-terminal region is protease sensitive and one region of the prion protein protease, designated as PrP 27-30, is resistant is.
Bei herkömmlichen Tests wird daher die zu untersuchende Probe zunächst in einem ersten Schritt (Schritt a) mit einer Protease verdaut, wobei davon ausgegangen wird, daß nach Verdauung in der Probe keine Protease sensitiven Bereiche des Prion-Proteins mehr enthalten sind und bei infektiösem Probematerial nur der Protease resistente Bereich PrP 27-30 der PrPSc-Form übrig bleibt. Dementsprechend wird nach der Verdauung in einem weiteren Schritt (Schritt b) lediglich überprüft, ob der Bereich PrP 27-30 in der Probe nachzuweisen ist oder nicht. Zum Nachweis werden z.B. spezifische im Bereich PrP 27-30 bindende Antikörper eingesetzt. Eventuell gebildete Antiköφer-PrP 27-30-Komplexe werden dann mittels herkömmlicher Methoden z.B. Elisa-Tests (Moynagh und Schim- mel;Nature 1999 Jul 8; 400 (6470): 105) nachgewiesen. Ist der Nachweis positiv, d.h. lassen sich z.B. Antiköφer-PrP 27-30 Komplexe nachweisen, so wird davon ausgegangen, daß die Probe PrPSc enthält und daß der Organismus, aus dem die Probe stammt, infiziert war.In conventional tests, the sample to be examined is therefore first digested with a protease in a first step (step a), it being assumed that after digestion, no protease-sensitive areas of the prion protein are present in the sample and only in the case of infectious sample material the protease resistant region PrP 27-30 of the PrP Sc form remains. Accordingly, after the digestion, in a further step (step b) it is only checked whether the area PrP 27-30 can be detected in the sample or not. For example, specific antibodies binding in the range PrP 27-30 are used for detection. Any antibody-PrP 27-30 complexes formed are then detected using conventional methods, for example Elisa tests (Moynagh and Schimmel; Nature 1999 Jul 8; 400 (6470): 105). If the proof is positive, ie if antibody-PrP 27-30 complexes can be detected, it is assumed that the sample contains PrP Sc and that the organism from which the sample comes was infected.
Ein Problem bei den herkömmlichen Tests ist, daß sie mit einem indirekten Nachweis arbeiten. Mit anderen Worten, aus der Tatsache, daß nach der Verdauung ggf. noch PrP 27-30 nachweisbar ist, wird geschlossen, daß es sich um den entsprechenden Protease resistenten Bereich aus PrPSc handelt, ohne daß die eingesetzte Methode diesen sicher von einem aus PrPc stammenden Bereich differenzieren kann. Unter ungünstigen Umständen, z.B. bei schwierig zu bearbeitendem Probematerial, kann es theoretisch zu falsch positiven Ergebnissen kommen.A problem with conventional tests is that they work with indirect detection. In other words, from the fact that after digestion, PrP 27-30 may still be detectable, it is concluded that it is the corresponding protease-resistant region from PrP Sc , without the method used being safe from a PrP c differentiate originating area. Under unfavorable circumstances, for example with sample material that is difficult to process, theoretically false positive results can occur.
Aufgabe der Erfindung ist es, gattungsgemäße Verfahren so weiterzubilden, daß eine sicherere Aussage möglich ist.The object of the invention is to develop generic methods so that a more reliable statement is possible.
Gelöst wird die Aufgabe mit einem Verfahren, das die kennzeichnenden Merkmale des Anspruches 1 aufweist.The object is achieved with a method which has the characterizing features of claim 1.
Bei dem erfindungsgemäßen Verfahren ist vorgesehen, daß nach dem Verdauungsschritt (Schritt a) die Probe in Schritt b nicht nur darauf übeφrüft wird, ob in ihr der Bereich PrP 27-30 enthalten ist, sondern zusätzlich geprüft wird, ob in der Probe noch Protease-sensitive Bereiche des Prion-Proteins enthalten sind.In the method according to the invention it is provided that after the digestion step (step a) the sample in step b is not only checked to determine whether it contains the PrP 27-30 region, but also to check whether protease is still present in the sample. sensitive areas of the prion protein are contained.
Das erfindungsgemäße Verfahren erlaubt damit, gleichzeitig eine Aussage über das eventuelle Vor- bzw. Nichtvorliegen von PrP 27-30 in der verdauten Probe und eine Aussage darüber, ob die Verdauung vollständig war oder nicht.The method according to the invention thus allows a statement about the possible presence or absence of PrP 27-30 in the digested sample and a statement as to whether the digestion was complete or not.
Der positive Nachweis von PrP 27-30 in einer verdauten Probe wird nur dann als Hinweis auf PrPSc gewertet, wenn gleichzeitig in der verdauten Probe keinerlei Protease sensitive Bereiche des Prion-Proteins mehr nachweisbar sind. Lassen sich solche Protease sensitiven Bereiche auch nach Verdauung noch in der Probe nachweisen, so ist der eventuelle Nachweis von PrP 27-30 kein eindeutiger Hinweis auf PrPSc sondern könnte auch bedeuten, daß ein entsprechende Bereich der PrPc-Form nicht vollständig verdaut war. In diesem Fall müßte die Probe noch einmal untersucht werden mit z.B. höheren Proteasekonzentrationen oder längeren Verdauungszeiten.The positive detection of PrP 27-30 in a digested sample is only considered as an indication of PrP Sc if none of the digested sample is present at the same time Protease sensitive areas of the prion protein are more detectable. If such protease sensitive areas can still be detected in the sample even after digestion, the possible detection of PrP 27-30 is not a clear indication of PrP Sc but could also mean that a corresponding area of the PrP c form was not completely digested. In this case the sample would have to be examined again with, for example, higher protease concentrations or longer digestion times.
Mit dem erfindungsgemäßen Verfahren lassen sich also falsch positive Untersuchungsergebnisse in besonders sicherer und einfacher Weise ausschließen. Gerade bei seltenen Infektionskrankheiten wie Prionenerkrankungen hängt die Aussagekraft eines Tests wesentlich davon ab, daß falsch positive Resultate minimiert werden.With the method according to the invention, false positive examination results can be excluded in a particularly safe and simple manner. Especially in the case of rare infectious diseases such as prion diseases, the significance of a test essentially depends on the fact that false positive results are minimized.
In einer bevorzugten Ausgestaltung der Erfindung erfolgt der Nachweis von PrP 27-30 bzw. Protease sensitiver Bereiche des Prion-Proteins mittels spezifisch in den jeweiligen Bereichen am Prion-Protein-bindenden Molekülen, die im folgenden als Molekül A (spezifisch für einen Protease-sensitiven Bereich) und Molekül B (spezifisch für den Bereich PrP 27-30) bezeichnet werden sollen.In a preferred embodiment of the invention, PrP 27-30 or protease-sensitive regions of the prion protein are detected by means of molecules which bind specifically to the prion protein in the respective regions and which are described below as molecule A (specific for a protease-sensitive) Area) and molecule B (specifically for the area PrP 27-30).
Ein übliches Verfahren gemäß dieser Ausgestaltung sieht vor, daß in Schritt a die Verdauung der Probe erfolgt und in Schritt b die Moleküle A und B der verdauten Probe zugesetzt werden und übeφrüft wird, ob sich in der Probe Komplexe aus dem Prion-Protein mit dem Molekül A und/oder B gebildet haben. In Abhängigkeit davon, ob und welche Komplexe sich gebildet haben, erfolgt dann die Auswertung.A common method according to this embodiment provides that in step a the digestion of the sample takes place and in step b the molecules A and B of the digested sample are added and it is checked whether there are complexes from the prion protein with the molecule in the sample A and / or B have formed. The evaluation is then carried out depending on whether and which complexes have formed.
Lassen sich nur Komplexe aus Molekül B und Prion-Protein nachweisen, so enthält die Probe PrPSc. Liegen zusätzlich Komplexe vor, die das Molekül A enthal- ten, so besteht die Gefahr eines falsch positiven Ergebnisses. Lassen sich keine Komplexe oder nur Komplexe mit Molekül A finden, so ist die Probe negativ.If only complexes of molecule B and prion protein can be detected, the sample contains PrP Sc . Are there additional complexes that contain the molecule A there is a risk of a false positive result. If no complexes or only complexes with molecule A are found, the sample is negative.
Als Moleküle A und B können insbesondere Antiköφer (im folgenden mit Anti- köφer A und B bezeichnet), die spezifisch die entsprechenden Bereiche des Prion-Proteins erkennen, eingesetzt werden. Genauso gut ist es aber auch möglich, daß andere spezifisch bindende Moleküle z.B. RNA-Moleküle eingesetzt werden.Molecules A and B can in particular be antibodies (hereinafter referred to as antibodies A and B) which specifically recognize the corresponding regions of the prion protein. However, it is equally possible that other specifically binding molecules e.g. RNA molecules are used.
Antikörper, die PrP 27-30 erkennen, sind ausfuhrlich beschrieben und dokumentiert. Es wird daher an dieser Stelle nicht näher darauf eingegangen.Antibodies that recognize PrP 27-30 are described and documented in detail. It is therefore not discussed in more detail here.
Antikörper die den Protease-sensitiven N-terminalen Bereich des PrP erkennen sind z.B. aus "Brain Research, 545, (1991) 319-321 (Antiserum anti-PrP-N), "Brain Pathol. 2002; 12: 1-11 (Antikörper FH11, BG4)", "Proc. Natl. Acad. Sei. Vol. 95 pp. 8812-8815, Juli 1998 (Antiköφer 5B2)M oder "Biochemical and Bio- physical Research Communications 273, 136-139 (2000) (Antiköφer 8B4)" bekannt. Die genannten Veröffentlichungen beschreiben die Eigenschaften der Antiköφer sowie ihre Herstellung.Antibodies which recognize the protease-sensitive N-terminal region of the PrP are, for example, from "Brain Research, 545, (1991) 319-321 (antiserum anti-PrP-N)," Brain Pathol. 2002; 12: 1-11 (Antibodies FH11, BG4) "," Proc. Natl. Acad. Be. Vol. 95 pp. 8812-8815, July 1998 (Antiköφer 5B2) M or "Biochemical and Biophysical Research Communications 273, 136-139 (2000) (Antiköφer 8B4)". The publications mentioned describe the properties of the antibodies and their production.
Der Nachweis der gebildeten Komplexe kann standardmäßig erfolgen. Zumeist ist vorgesehen, daß eine der beiden Komponenten des gebildeten Komplexes trägergebunden ist.The complexes formed can be verified as standard. It is usually provided that one of the two components of the complex formed is carrier-bound.
Denkbar ist z.B., daß das Probematerial nach Verdauung z.B. an einer Mikroti- terplatte oder an Beads immobilisiert wird und eine Detektion mit markierten Molekülen A und B, insbesondere Antiköφern A und B, vorgenommen wird. Die vorzugsweise eingesetzten Antiköφer können in einem Ansatz gleichzeitig oder nacheinander mit dem Probematerial inkubiert werden. Genausogut ist es möglich zwei parallele Ansätze durchzuführen, in denen jeweils einer der beiden Antikörper A oder B zugesetzt wird.It is conceivable, for example, that the sample material is immobilized after digestion, for example on a microtiter plate or on beads, and a detection with labeled molecules A and B, in particular antibodies A and B, is carried out. The preferably used antibodies can be incubated in one batch simultaneously or in succession with the sample material. It is equally possible to carry out two parallel runs, in each of which one of the two antibodies A or B is added.
Denkbar ist natürlich auch, die Moleküle A und B, bevorzugt die Antiköφer A und B, jeweils auf Chips zu immobilisieren, die in Abhängigkeit von einer an ihrer Oberfläche erfolgenden molekularen Interaktion ein meßbares Signal erzeugen. Solche Chips sind aus der EP 887645 bekannt. Werden solche Chips, an die jeweils z.B Antiköφer A bzw B immobilisiert sind, mit dem nach Verdauung erhaltenen Probematerial inkubiert, so läßt sich auf einfache Weise messen, z.B. anhand der optischen Lichtbrechung, ob das Probematerial von den auf den Chips immobilisierten Antiköpern gebunden wurde oder nicht.It is of course also conceivable to immobilize the molecules A and B, preferably the antibodies A and B, on chips which generate a measurable signal depending on a molecular interaction taking place on their surface. Such chips are known from EP 887645. If such chips, to which antibodies A or B are immobilized, for example, are incubated with the sample material obtained after digestion, it can be measured in a simple manner, e.g. based on the optical light refraction, whether or not the sample material was bound by the antibodies immobilized on the chips.
Bevorzugt wird zum Nachweis ein Sandwichimmunoassay durchgeführt. Grundsätzlich kommen bei solchen Sandwichimmunoassays jeweils zwei Antiköφer pro Analyt zum Einsatz, die an zwei unterschiedliche Epitope des Analyten gebunden werden. Einer der Antiköφer ist in der Regel immobilisiert und dient zur Kopplung des Analyten an eine Festphase, während der andere als Detektionsan- tiköφer dient und mit einer Markierung versehen ist.A sandwich immunoassay is preferably carried out for detection. In principle, two antibodies per analyte are used in such sandwich immunoassays, which are bound to two different epitopes of the analyte. One of the antibodies is usually immobilized and serves to couple the analyte to a solid phase, while the other serves as a detection antibody and is provided with a label.
Im vorliegenden Fall wird in diesem Zusammenhang neben den Antiköφern A und B, die die unterschiedlichen Bereiche des PrP erkennen, zusätzlich noch ein weiterer Antiköφer C vorgesehen, der PrP 27-30 erkennt, wobei allerdings das von diesem Antiköφer C erkannte Epitop ein anderes ist als das von Antiköφer B erkannte. Es gibt nun eine Reihe unterschiedlicher Möglichkeiten:In the present case, in addition to the antibodies A and B, which recognize the different areas of the PrP, a further antibody C is also provided in this context, which recognizes PrP 27-30, although the epitope recognized by this antibody C is different than that recognized by Antiköφer B. There are now a number of different options:
Denkbar ist Antiköφer C auf einem Träger zu immobilisieren, den Träger dann mit dem nach Verdauung erhaltenen Probematerial zu inkubieren und dann zur Detektion markierte Antikörper A und B zuzusetzen.It is conceivable to immobilize Antibody C on a support, then incubate the support with the sample material obtained after digestion and then add labeled antibodies A and B for detection.
Eine andere Möglichkeit ist, die Antiköφer A und B auf einem Träger zu immobilisieren und dann nach Inkubation des Trägers mit dem Probematerial zur Detektion markiertem Antiköφer C zuzusetzen.Another possibility is to immobilize the antibodies A and B on a support and then, after incubation of the support with the sample material, to add labeled antibody C for detection.
Im Hinblick auf die erforderliche Auflösung der Signale, auf eine Standardisierung und auch generell im Hinblick auf Probleme, die mit einer Triplekinetik einhergehen, könnten die beiden letztgenannten Varianten mit Problemen verbunden sein.With regard to the required resolution of the signals, a standardization and also generally with regard to problems associated with triple kinetics, the latter two variants could be associated with problems.
Diesen Problemen kann man dadurch begegnen, daß man die Reaktionen trennt, z.B., daß man die Antiköφer auf unterschiedlichen Trägern immobilisiert und die Inkubationen in unterschiedlichen Ansätzen durchführt.These problems can be countered by separating the reactions, e.g. by immobilizing the antibodies on different supports and carrying out the incubations in different approaches.
Denkbar ist gemäß einer besonders bevozugten Ausgestaltung aber auch, daß das nach Verdauung erhaltene Probematerial in einem Ansatz zuerst mit immobilisierten Antikörpern A und dann mit immobilisierten Antiköφern B inkubiert wird. Zur Detektion wird wie oben beschrieben markierter Antiköφer C zugesetzt. Bei dieser Ausgestaltung erreicht man durch die zeitliche Staffelung auf einfache Weise, daß eventuell vorhandene Protease-sentitive Bereiche des PrP zunächst an die spezifischen Antiköφer A binden können, ohne daß die Bindungskinetik durch einen gleichzeitigen Angriff der als Molekül B dienenden Antikörper auf den Protease-resistenten Bereich gestört wird. Denkbar ist z.B. die Probe nacheinander mit Beads zu versetzen, die mit den betreffenden Antiköφern markiert sind, oder den Ansatz mit einer Einrichtung durchzuführen, bei der das Probematerial im Durchfluß nacheinander mit räumlich getrennten Bereichen in der Einrichtung kontaktiert wird, in denen jeweils die Antikörper A bzw. B immobilisiert sind.However, according to a particularly preferred embodiment, it is also conceivable that the sample material obtained after digestion is incubated in one batch first with immobilized antibodies A and then with immobilized antibodies B. Labeled antibody C is added for detection as described above. In this embodiment, the staggering over time makes it possible in a simple manner that any protease-sensitive areas of the PrP that are present can initially bind to the specific antibodies A, without the binding kinetics being caused by a simultaneous attack by the antibodies serving as molecule B on the protease-resistant Area is disturbed. It is conceivable, for example, to place the sample in succession with beads which are labeled with the antibodies in question, or to carry out the approach with a device in which the sample material is successively contacted in the flow with spatially separated areas in the device, in each of which the antibodies A or B are immobilized.
Wie oben angesprochen, erfolgt der Nachweis eventuell gebildeter Komplexe mittels markierter Moleküle, insbesondere markierter Antiköφer. Läßt sich auf einem Träger eine Markierung nachweisen bzw. erkennen, so ist dies ein Zeichen, daß der betreffende mit der nachgewiesen Markierung versehene Antikörper gebunden wurde, was in Abhängigkeit von dem Testaufbau ein Indiz für das Vorliegen eines bestimmten Komplexes gewertet werden kann.As mentioned above, any complexes formed are detected using labeled molecules, in particular labeled antibodies. If a label can be detected or recognized on a support, this is a sign that the relevant antibody provided with the detected label has been bound, which, depending on the test setup, can be an indication of the presence of a certain complex.
Die Moleküle A und B, bzw der Antiköφer C können mit z.B. gleichen oder unterschiedlichen Fluoreszenzmarkierungen oder mit Enzymen (Elisa) bzw. mit anderen geeigneten Markierungen markiert sein. Grundsätzlich sind alle Markierungen geeignet, die sich direkt oder indirekt nachweisen bzw. messen lassen. Die unterschiedlichen Möglichkeiten, Moleküle insbesondere Antiköφer in geeigneter Weise für obige Verfahren zu markieren und im Rahmen der Verfahren nachzuweisen sind dem Fachmann bekannt. Es soll daher an dieser Stelle nicht näher darauf eingegangen werden The molecules A and B or the antibody C can be e.g. the same or different fluorescent labels or with enzymes (Elisa) or with other suitable labels. Basically, all markings are suitable that can be directly or indirectly detected or measured. The person skilled in the art is aware of the different possibilities for labeling molecules, in particular antibodies, in a suitable manner for the above processes and for detecting them in the course of the processes. It should therefore not be dealt with in more detail at this point

Claims

PATENTANSPRÜCHE
Verfahren zur Untersuchung von Prion-Protein enthaltenden Proben auf das eventuelle Vorliegen der PrPSc-Form, bei demMethod for examining samples containing prion protein for the possible presence of the PrP Sc form, in which
a) die Probe mit Protease versetzt wird zur Verdauung Protease sensitiver Proteine bzw. Proteinbereiche,a) protease is added to the sample for digestion of protease-sensitive proteins or protein regions,
b) die Probe nach Verdauung darauf übeφrüft wird, ob in ihr der Bereich PrP 27-30 des Prion-Proteins enthalten ist, der in der PrPSc-Form des Prion-Proteins Protease resistent ist undb) after digestion, the sample is checked to determine whether it contains the PrP 27-30 region of the prion protein which is resistant in the PrP Sc form of the prion protein protease and
c) aus dem positiven Nachweis von PrP 27-30 auf das Vorliegen von PrPSc in der Probe geschlossen wird,c) the presence of PrP Sc in the sample is concluded from the positive detection of PrP 27-30,
dadurch gekennzeichnet, daß im Schritt b) die Probe zusätzlich darauf überprüft wird, ob eine Verdauung des Protease sensitiven Bereichs des Prion-Proteins stattgefunden hat.characterized in that in step b) the sample is additionally checked to see whether the protease-sensitive region of the prion protein has been digested.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß im Schritt b) die Probe mit an das Prion-Protein bindenden Molekülen A und B versetzt wird, wobei Molekül A in einem Protease sensitiven Bereich des PrP- Proteins bindet, und Molekül B im Bereich PrP 27-30 bindet, und eventuell in der Probe gebildete Komplexe aus Prion-Protein und den Molekülen A und/oder B nachgewiesen werden.2. The method according to claim 1, characterized in that in step b) the sample with the prion protein binding molecules A and B is added, wherein molecule A binds in a protease sensitive region of the PrP protein, and molecule B in the region PrP 27-30 binds, and any complexes of prion protein and molecules A and / or B formed in the sample can be detected.
3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß die in Schritt b) eingesetzten Moleküle A und/oder B Antiköφer sind. 3. The method according to claim 2, characterized in that the molecules A and / or B used in step b) are Antiköφer.
4. Verfahren nach ein Anspruch 3, dadurch gekennzeichnet, daß der Nachweis der gebildeten Komplexe aus Prion-Protein und den Molekülen A und B in einem Sandwichimmunoassay erfolgt.4. The method according to claim 3, characterized in that the detection of the complexes formed from prion protein and the molecules A and B is carried out in a sandwich immunoassay.
5. Verfahren nach Anspruch 4, dadurch gekennzeichnet, daß die Probe nach Verdauung zuerst mit immobilisierten als Moleküle A dienenden Antiköφern, dann mit immobilisierten als Moleküle B dienenden Antikörpern in Kontakt gebracht wird, und dann mit einem markiertem PrP 27-30 erkennenden Antiköφer eventuell gebildete Komplexe aus Prion-Protein und immobilisierten Antiköφer nachgewiesen werden.5. The method according to claim 4, characterized in that after digestion, the sample is first brought into contact with immobilized antibodies serving as molecules A, then with immobilized antibodies serving as molecules B, and then possibly formed with a labeled PrP 27-30-recognizing antibody Complexes of prion protein and immobilized antibodies can be detected.
6. Verfahren nach einem der Ansprüche 2-4, dadurch gekennzeichnet, daß im Schritt b) zunächst die Probe in zwei Ansätze aufgeteilt wird und jedem der Ansätze dann jeweils die Moleküle A oder B zugesetzt werden. 6. The method according to any one of claims 2-4, characterized in that in step b) the sample is first divided into two batches and each of the batches then the molecules A or B are added.
EP02737971A 2001-04-21 2002-04-19 Method for testing samples containing prion protein for the possible presence of the prp?sc form Withdrawn EP1381868A2 (en)

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DE10119713A DE10119713A1 (en) 2001-04-21 2001-04-21 Testing samples for the presence of pathological prions, useful for detecting e.g. bovine spongiform encephalopathy, based on differential sensitivity to proteases
PCT/EP2002/004341 WO2002086511A2 (en) 2001-04-21 2002-04-19 Method for testing samples containing prion protein for the possible presence of the prpsc form

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FR2849205B1 (en) * 2002-12-20 2005-02-11 Afssa METHOD FOR AMPLIFYING PRPSC DETECTION AND USE OF A MACROCYCLIC ADJUVANT LIGAND FOR SUCH AMPLIFICATION
FR2849204B1 (en) * 2002-12-20 2005-02-11 Afssa METHOD OF DETECTING PRPSC USING AMINOGLYCOSIDE FAMILY D Antibiotics for PRPSC Removal and Detection in Biological Samples
FR2865280B1 (en) 2004-01-20 2007-01-12 Biomerieux Sa METHOD OF DETECTING PRP USING MOLECULE HAVING AT LEAST ONE POSITIVE LOAD AND / OR AT LEAST ONE OSIDIC BOND AND LIGAND OTHER THAN A PROTEIN LIGAND
EP1596199A1 (en) * 2004-05-14 2005-11-16 Prionics AG Method for the detection of disease-related prion
EP1677115B1 (en) * 2004-11-15 2008-03-12 Roche Diagnostics GmbH High-throughput prion assays
FR2888937B1 (en) 2005-07-21 2012-10-26 Biomerieux Sa METHOD OF DETECTING FCPA USING FCPA AGGREGATION AGENT AND FORM AGGREGATE CAPTURING AGENT
DE102007016324A1 (en) * 2007-04-04 2008-10-09 Priontype Gmbh & Co.Kg Method for the detection of pathologically altered prion protein (PrPSc)
WO2010084201A1 (en) 2009-01-26 2010-07-29 Commissariat A L'energie Atomique Et Aux Energies Alternatives Novel derivative of erythromycin for the treatment and diagnosis of prion disease

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AU738606B2 (en) * 1997-02-06 2001-09-20 Enfer Technology Limited Immunological assay for spongiform encephalopathies
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FI982481A0 (en) * 1998-11-17 1998-11-17 Wallac Oy Immunoassay for the detection of infectious bovine spongiform encephalopathy
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