WO2002066987A2 - Method for the determination of specific proteins and an application - Google Patents
Method for the determination of specific proteins and an application Download PDFInfo
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- WO2002066987A2 WO2002066987A2 PCT/EP2002/001303 EP0201303W WO02066987A2 WO 2002066987 A2 WO2002066987 A2 WO 2002066987A2 EP 0201303 W EP0201303 W EP 0201303W WO 02066987 A2 WO02066987 A2 WO 02066987A2
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- specific protein
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 38
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 28
- 208000024777 Prion disease Diseases 0.000 claims abstract description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 13
- 201000010099 disease Diseases 0.000 claims abstract description 12
- 208000018756 Variant Creutzfeldt-Jakob disease Diseases 0.000 claims abstract description 10
- 208000005881 bovine spongiform encephalopathy Diseases 0.000 claims abstract description 10
- 208000008864 scrapie Diseases 0.000 claims abstract description 6
- 208000020406 Creutzfeldt Jacob disease Diseases 0.000 claims abstract description 5
- 208000003407 Creutzfeldt-Jakob Syndrome Diseases 0.000 claims abstract description 5
- 208000010859 Creutzfeldt-Jakob disease Diseases 0.000 claims abstract description 5
- 206010023497 kuru Diseases 0.000 claims abstract description 5
- 238000001514 detection method Methods 0.000 claims description 17
- 241001465754 Metazoa Species 0.000 claims description 15
- 241000283690 Bos taurus Species 0.000 claims description 8
- 210000001742 aqueous humor Anatomy 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 210000001525 retina Anatomy 0.000 claims description 5
- 102000001708 Protein Isoforms Human genes 0.000 claims description 4
- 108010029485 Protein Isoforms Proteins 0.000 claims description 4
- 239000013543 active substance Substances 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 238000000760 immunoelectrophoresis Methods 0.000 claims description 3
- 210000004127 vitreous body Anatomy 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 19
- 210000001508 eye Anatomy 0.000 description 17
- 210000004556 brain Anatomy 0.000 description 9
- 102000029797 Prion Human genes 0.000 description 4
- 108091000054 Prion Proteins 0.000 description 4
- 210000002159 anterior chamber Anatomy 0.000 description 4
- 210000004087 cornea Anatomy 0.000 description 4
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 210000003786 sclera Anatomy 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 101710177166 Phosphoprotein Proteins 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 210000003128 head Anatomy 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 208000037259 Amyloid Plaque Diseases 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 210000003423 ankle Anatomy 0.000 description 1
- 230000002358 autolytic effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000005252 bulbus oculi Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002695 general anesthesia Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003589 local anesthetic agent Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000000944 nerve tissue Anatomy 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
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
- 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 for the detection of specific proteins which are associated with diseases such as transmissible spongiform encephalopathies (TSE), bovine spongiform encephalopathy (BSE), Kuru, scrapie, Creutzfeldt-Jakob disease and the like. It also relates to use.
- TSE transmissible spongiform encephalopathies
- BSE bovine spongiform encephalopathy
- Kuru scrapie
- Creutzfeldt-Jakob disease Creutzfeldt-Jakob disease and the like. It also relates to use.
- spongiform encephalopathies are diseases that are associated with the appearance and deposition of specific pathogenic proteins (PK).
- PK pathogenic proteins
- P and PK can be so-called prion proteins.
- PK differs in particular from P. in its spatial structure has a ⁇ -sheet structure. It has an increased tendency to aggregate and is more resistant to proteases than the physiological protein (CP). PK accumulates in the nerve tissue. There it forms so-called amyloid deposits, which are made responsible for the symptoms of the disease.
- a brain sample is taken to produce a sample for the detection, for example, of the pathological prion protein PrP sc .
- the brain sample is then digested enzymatically to destroy PrP c and separate it from PrP sc .
- the remaining undigested PrP sc is then detected by means of enzyme immunological methods or the immunoblot technique.
- the preparation of the sample is time-consuming and costly. In addition, it is necessary to quickly transport the brain sample to the examination facility because its decomposition can lead to a falsification of the PrP sc detection.
- the enzymatic preparation of the brain sample is also time-consuming. Costly devices are required to carry them out.
- the object of the invention is to eliminate the disadvantages of the prior art.
- a method and a use are to be specified which enable simple and rapid detection of diseases associated with specific proteins, such as transmissible spongiform encephalopathies.
- the method according to the invention enables simple and rapid detection of diseases associated with specific proteins, such as transmissible spongiform encephalopathies.
- a portion of the eye, such as aqueous humor, can also be removed from the live animal with a small intervention. It is not absolutely necessary to kill the animal in order to carry out the detection procedure for the presence of specific prion proteins. It is also much easier to borrow parts of the eye from the slaughtered animal than to take a brain sample.
- a removed eye is protected against microbial contamination for some time by the cornea and sclera. In contrast to a brain test, decomposition and autolytic processes occur much later.
- c can be a conventional detection method for pathological prion proteins.
- sample solution produced using eye portions can be examined for the presence of specific proteins by means of a conventional detection method without the lengthy pretreatments which would be required for the starting material brain.
- PrP sc for the detection of PrP sc is / are used as an eye portion, preferably aqueous humor and / or
- Vitreous and / or the retina used The aqueous humor in particular can also be taken from living animals. All that is required is a minor veterinary intervention. Living breeding animals, zoo or wild animals, which are particularly difficult to replace, can be spared in this way; they do not have to be killed to demonstrate the presence of PrP sc . Furthermore, the vitreous, the aqueous humor and the retina are almost free of disturbing protein and pias minogen. Compared to using a brain sample, lengthy cleaning and inactivation steps are eliminated.
- the eye parts are removed from the eye of an animal, in particular a cattle.
- the method according to the invention is particularly well suited for cattle, because protein synthesis is also physiologically possible in the cattle eye.
- it is possible to concentrate the specific protein PK, in particular PrP sc from the sample by means of gradient centrifugation or capillary immuno-electrophoresis. It is also possible to use affinity processes for concentration without purification or dialysis processes with a desalination step.
- a proteolytically active substance can be added to the sample which, under defined reaction conditions, leaves the specific protein PK essentially unaffected and cleaves a protein P which forms an isoform of PK.
- the defined reaction conditions are reaction conditions which are similar to physiological conditions.
- the specific protein Pk can be PrP sc and the protein P can be PrP c .
- An enzyme, for example proteinase-K, is expediently used as the proteolytically active substance.
- an antibody which binds to the specific protein PK preferably labeled, can be added in a conventional manner.
- TSE transmissible spongiform encephalopathies
- BSE bovine spongiform phalopathy
- Kuru scrapie
- Creutzfeldt-Jakob disease and the like.
- a sample from the dead toer e.g.
- One or both eyes were removed from a slaughtered cattle after the skull had been removed using the usual technique.
- the eye (s) are placed in sterile transport containers and labeled in such a way that they can be clearly assigned to the associated carcass.
- the sample material is then cooled.
- the device After disinfecting the surface of the eye at the outermost edge of the cornea at the transition to the sclera, the device is inserted using sterile injection equipment.
- the injection needle is inserted into the anterior chamber parallel to the iris and the aqueous humor in the anterior chamber is aspirated until the anterior chamber has completely collapsed.
- the eyeball is split in half with a sterile scalpel and the gelatinous vitreous is removed with sterile cutlery (tweezers, sharp spoons, etc.).
- sterile cutlery tweezers, sharp spoons, etc.
- All eye parts obtained in this way are placed in separate sterile sample vessels for further processing.
- a cattle is first sedated to obtain a sample from the living animal.
- the animal can also be put under general anesthesia if it is an animal not used for food production or a very shy (zoo) animal.
- general coercive measures ankle cuffs, head rope
- a local anesthetic is dripped onto the cornea of the eye to be examined and the rest of the head is covered.
- a sterile induction set is inserted at the temporal edge of the cornea at the transition to the sclera, the injection needle is inserted into the anterior chamber of the eye parallel to the course of the iris and aspirated aqueous humor.
- the parts of the eye removed from the living or dead animal are subsequently mechanically or physically comminuted and thus homogenized to produce the sample.
- the comminution can e.g. by means of ultrasound or a high-speed agitator, e.g. an Ultrathurrax.
- the homogenized liquid produced can subsequently be filtered or centrifuged.
- the sample can then be obtained from the supernatant.
- the sample can be examined directly for the presence of PrP sc using conventional methods.
- the lengthy preparation process currently used according to the prior art of a sample taken from the brain is eliminated.
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- Urology & Nephrology (AREA)
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Abstract
Description
Beschreibungdescription
Verfahren zum Nachweis spezifischer Proteine und VerwendungMethod for detection of specific proteins and use
Die Erfindung betrifft ein Verfahren zum Nachweis spezifischer Proteine, welche mit Krankheiten, wie transmissible spongiforme Enzephalopathien (TSE) , bovine spongiforme Enze- phalopathie (BSE) , Kuru, Scrapie, Creutzfeldt-Jakob-Krankheit, und dgl.. assoziiert sind. Sie betrifft ferner eine Verwendung .The invention relates to a method for the detection of specific proteins which are associated with diseases such as transmissible spongiform encephalopathies (TSE), bovine spongiform encephalopathy (BSE), Kuru, scrapie, Creutzfeldt-Jakob disease and the like. It also relates to use.
Bei den sogenannten spongiformen Enzephalopathien handelt es sich um Krankheiten, die mit dem Auftreten und der Ablagerung spezifischer pathogener Proteine (PK) assoziiert sind. Es handelt es sich bei den krankheitsspezifischen Proteinen umThe so-called spongiform encephalopathies are diseases that are associated with the appearance and deposition of specific pathogenic proteins (PK). The disease-specific proteins are
Isoformen physiologisch vorkommender Proteine (P) . Dabei können P und PK sogenannte Prionproteine sein. PK unterscheidet sich insbesondere in seiner räumlichen Struktur von P. PK besitzt eine ß-Faltblattstruktur . Es hat eine erhöhte Aggrega- tionsneigung und weist eine erhöhte Proteaseresistenz als das physiologische Protein (CP) auf. PK reichert sich im Nervengewebe an. Es bildet dort sogenannte amyloide Ablagerungen, welche für die KrankheitsSymptome mitverantwortlich gemacht werden .Isoforms of physiologically occurring proteins (P). P and PK can be so-called prion proteins. PK differs in particular from P. in its spatial structure has a β-sheet structure. It has an increased tendency to aggregate and is more resistant to proteases than the physiological protein (CP). PK accumulates in the nerve tissue. There it forms so-called amyloid deposits, which are made responsible for the symptoms of the disease.
Wodurch das Auftreten von PK hervorgerufen wird, ist bisher nicht bekannt. Sicher scheint allerdings zu sein, daß PK mitverantwortlich für die bei der Krankheit auftretenden Symptome ist. Das im Zusammenhang mit spongiformen Enzephalopathien beim Tier auftretende pathogene Prionprotein "Scrapie" wird im folgenden mit PrPsc, das physiologische Protein mit PrPc abgekürzt . Insbesondere im Hinblick auf die in Europa auftretende Rinderseuche BSE und der damit einhergehenden Gefahr einer Übertragung dieser Krankheit auf den Menschen, besteht ein hoher Bedarf an der Diagnose transmissibler spongiformer Enzephalo- pathien.It is not yet known what causes PK to occur. It seems certain, however, that PK is jointly responsible for the symptoms occurring with the disease. The pathogenic prion protein "scrapie" which occurs in connection with spongiform encephalopathies in the animal is abbreviated in the following with PrP sc , the physiological protein with PrP c . There is a great need for the diagnosis of transmissible spongiform encephalopathies, in particular with regard to the BSE disease that occurs in Europe and the associated risk of transmission of this disease to humans.
Zur Herstellung einer Probe zum Nachweis z.B. des pathologischen Prionproteins PrPsc wird nach dem Stand der Technik eine Gehirnprobe entnommen. Die Gehirnprobe wird dann enzyma- tisch verdaut, um PrPc zu zerstören und von PrPsc zu trennen. Das verbleibende nicht verdaute PrPsc wird dann mittels enzymimmunologischer Verfahren oder der Immunoblot-Technik nachgewiesen.According to the prior art, a brain sample is taken to produce a sample for the detection, for example, of the pathological prion protein PrP sc . The brain sample is then digested enzymatically to destroy PrP c and separate it from PrP sc . The remaining undigested PrP sc is then detected by means of enzyme immunological methods or the immunoblot technique.
Die Herstellung der Probe ist zeit- und kostenaufwendig. Außerdem ist es erforderlich, die Gehirnprobe schnell zur Untersuchungseinrichtung zu transportieren, weil deren Zersetzung zu einer Verfälschung des Nachweises von PrPsc führen kann. Die enzymatische Aufbereitung der Gehirnprobe ist eben- falls zeitaufwendig. Zu deren Durchführung sind kostenaufwendige Vorrichtungen erforderlich.The preparation of the sample is time-consuming and costly. In addition, it is necessary to quickly transport the brain sample to the examination facility because its decomposition can lead to a falsification of the PrP sc detection. The enzymatic preparation of the brain sample is also time-consuming. Costly devices are required to carry them out.
Aufgabe der Erfindung ist es, die Nachteile nach dem Stand der Technik zu beseitigen. Es sollen insbesondere ein Verfah- ren und eine Verwendung angegeben werden, welche einen einfachen und schnellen Nachweis von mit spezifischen Proteinen assoziierten Krankheiten, wie transmissible spongiforme Enzephalopathien, ermöglichen.The object of the invention is to eliminate the disadvantages of the prior art. In particular, a method and a use are to be specified which enable simple and rapid detection of diseases associated with specific proteins, such as transmissible spongiform encephalopathies.
Diese Aufgabe wird durch die Merkmale der Ansprüche 1 und 8 gelöst. Zweckmäßige Ausgestaltung ergeben sich aus den Merkmalen der Ansprüche 2 bis 7 und 9 bis 14. Das erfindungsgemäße Verfahren ermöglicht einen einfachen und schnellen Nachweis von mit spezifischen Proteinen assoziierten Krankheiten, wie transmissible spongiforme Enzephalopathien. Ein Anteil des Auges, wie Kammerwasser kann mittels eines kleinen Eingriffs auch vom lebenden Tier entnommen werden. Es ist nicht zwangsläufig erforderlich, zur Durchführung des Nachweisverfahrens auf das Vorliegen spezifischer Prion- proteine das Tier zu töten. Auch die Entnahme von Anteilen des Auges vom geschlachteten Tier ist deutlich einfacher mög- lieh als die Entnahme einer Gehirnprobe. Ein entnommenes Auge ist durch Kornea und Sklera vor mikrobieller Kontamination einige Zeit geschützt. Eine Zersetzung und autolytische Prozesse treten im Gegensatz zu einer Gehirnprobe wesentlich später auf. - Bei dem Nachweisverfahren gemäß Schritt lit. c kann es sich um ein herkömmliches Nachweisverfahren für pathologische Prionproteine handeln.This object is solved by the features of claims 1 and 8. A practical embodiment results from the features of claims 2 to 7 and 9 to 14. The method according to the invention enables simple and rapid detection of diseases associated with specific proteins, such as transmissible spongiform encephalopathies. A portion of the eye, such as aqueous humor, can also be removed from the live animal with a small intervention. It is not absolutely necessary to kill the animal in order to carry out the detection procedure for the presence of specific prion proteins. It is also much easier to borrow parts of the eye from the slaughtered animal than to take a brain sample. A removed eye is protected against microbial contamination for some time by the cornea and sclera. In contrast to a brain test, decomposition and autolytic processes occur much later. - In the detection procedure according to step lit. c can be a conventional detection method for pathological prion proteins.
Die unter Verwendung von Augenanteilen hergestellte Probenlösung kann ohne langwierige Vorbehandlungen, wie es das Aus- gangsmaterial Gehirn erfordern würde, mittels eines herkömmlichen Nachweisverfahrens auf das Vorhandensein von spezifischen Proteinen untersucht werden.The sample solution produced using eye portions can be examined for the presence of specific proteins by means of a conventional detection method without the lengthy pretreatments which would be required for the starting material brain.
Nach einer Ausgestaltung wird/werden für den Nachweis von PrPsc als Augenanteil/e, vorzugsweise Kammerwasser und/oderAccording to one embodiment, for the detection of PrP sc is / are used as an eye portion, preferably aqueous humor and / or
Glaskörper und/oder die Retina verwendet . Insbesondere das Kammerwasser kann auch am lebenden Tier entnommen werden. Dazu ist lediglich ein geringfügiger tierärztlicher Eingriff notwendig. Insbesondere schwer ersetzbare lebende Zuchttiere, Zoo- oder Wildtiere können auf diese Weise geschont werden; sie müssen für den Nachweis des Vorliegens von PrPsc nicht getötet werden. Ferner sind der Glaskörper, das Kammerwasser sowie die Retina nahezu frei von störendem Protein und Pias- minogen. Im Vergleich zur Verwendung einer Gehirnprobe entfallen langwierige Reinigungs- und Inaktivierungsschritte .Vitreous and / or the retina used. The aqueous humor in particular can also be taken from living animals. All that is required is a minor veterinary intervention. Living breeding animals, zoo or wild animals, which are particularly difficult to replace, can be spared in this way; they do not have to be killed to demonstrate the presence of PrP sc . Furthermore, the vitreous, the aqueous humor and the retina are almost free of disturbing protein and pias minogen. Compared to using a brain sample, lengthy cleaning and inactivation steps are eliminated.
Nach einem weiteren Ausgestaltungsmerkmal werden die Augenan- teile dem Auge eines Tiers, insbesondere eines Rinds, entnommen. Insbesondere beim Rind eignet sich das erfindungsgemäße Verfahren besonders gut, weil im Rinderauge auch physiologischerweise eine Proteinsynthese möglich ist. Schließlich ist es möglich, das spezifische Protein PK, insbesondere PrPsc, aus der Probe mittels Gradientenzentrifugation oder Kapillar- Immuno-Elektrophorese zu konzentrieren. Ferner ist es möglich zur Konzentration Affinitätsverfahren ohne Aufreinigung oder Dialyseverfahren mit einem Entsalzungsschritt zu verwenden.According to a further design feature, the eye parts are removed from the eye of an animal, in particular a cattle. The method according to the invention is particularly well suited for cattle, because protein synthesis is also physiologically possible in the cattle eye. Finally, it is possible to concentrate the specific protein PK, in particular PrP sc , from the sample by means of gradient centrifugation or capillary immuno-electrophoresis. It is also possible to use affinity processes for concentration without purification or dialysis processes with a desalination step.
Der Probe kann eine proteolytisch wirksame Substanz zugesetzt werden, welche unter definierten Reaktionsbedingungen das spezifische Protein PK im wesentlichen unbeeinflußt läßt und ein eine Isoform von PK bildendes Protein P spaltet. Bei den definierten Reaktionsbedingungen handelt es sich um Reakti- onsbedingungen, welche physiologischen Bedingungen ähnlich sind. Bei dem spezifischen Protein Pk kann es sich um PrPsc und bei dem Protein P um PrPc handeln. Als proteolytisch wirksame Substanz wird zweckmäßigerweise ein Enzym, z.B. Pro- teinase-K, verwendet. Zum Nachweis des spezifischen Proteins PK kann in herkömmlicher Weise ein an das spezifische Protein PK bindender, vorzugsweise markierter, Antikörper zugesetzt werden.A proteolytically active substance can be added to the sample which, under defined reaction conditions, leaves the specific protein PK essentially unaffected and cleaves a protein P which forms an isoform of PK. The defined reaction conditions are reaction conditions which are similar to physiological conditions. The specific protein Pk can be PrP sc and the protein P can be PrP c . An enzyme, for example proteinase-K, is expediently used as the proteolytically active substance. To detect the specific protein PK, an antibody which binds to the specific protein PK, preferably labeled, can be added in a conventional manner.
Nach weiterer Maßgabe der Erfindung wird die Verwendung von Anteilen des Augens zur Herstellung einer Probe für dieAccording to a further aspect of the invention, the use of portions of the eye for the preparation of a sample for the
Durchführung eines Nachweisverfahrens auf spezifische Proteine PK beansprucht, welche mit Krankheiten, wie transmissible spongiforme Enzephalopathien (TSE) , bovine spongiforme Enze- phalopathie (BSE) , Kuru, Scrapie, Creutzfeldt-Jakob-Krankheit, und dgl. assoziiert sind.Carrying out a detection procedure for specific proteins PK, which is associated with diseases such as transmissible spongiform encephalopathies (TSE), bovine spongiform phalopathy (BSE), Kuru, scrapie, Creutzfeldt-Jakob disease, and the like.
Wegen der vorteilhaften Ausgestaltungen der Verwendung wird auf die vorangegangenen Ausführungen verwiesen. Nachfolgend wird die Erfindung anhand eines Ausführungsbeispiels näher erläutert .Because of the advantageous embodiments of the use, reference is made to the preceding explanations. The invention is explained in more detail below using an exemplary embodiment.
Zur Herstellung einer Probe vom toten Toer werden z.B. einem geschlachteten Rind nach Absetzen des Schädels ein oder beide Augen nach der üblichen Technik entnommen. Das Auge/die Augen werden in sterile Transportgefäße gegeben und diese so gekennzeichnet, daß sie eindeutig dem zugehörigen Schlachtkörper zuordenbar sind. Anschließend wird das Probenmaterial ge- kühlt.For the preparation of a sample from the dead toer, e.g. One or both eyes were removed from a slaughtered cattle after the skull had been removed using the usual technique. The eye (s) are placed in sterile transport containers and labeled in such a way that they can be clearly assigned to the associated carcass. The sample material is then cooled.
Mittels sterilem Injektionsbesteck wird nach Oberflächendesinfektion des Auges am äußersten Rand der Kornea am Übergang zur Sklera eingestochen. Die Injektionsnadel wird parallel zur Iris in die vordere Augenkammer eingebracht und das sich in der vorderen Augenkammer befindliche Kammerwasser aspiriert, bis die vordere Augenkammer gänzlich kollabiert ist. Anschließend wird der Augapfel mit einem sterilen Skalpell in zwei Hälften gespalten und mit einem sterilen Besteck (Pin- zette, scharfen Löffel etc.) der gelatinöse Glaskörper herausgelöst. Zuletzt wird die Retina, sofern sie nicht durch die hervorgehende Maßnahme mit abgelöst ist, vorsichtig von den restlichen Augenanteilen getrennt und mit eienr Pinzette abgelöst .After disinfecting the surface of the eye at the outermost edge of the cornea at the transition to the sclera, the device is inserted using sterile injection equipment. The injection needle is inserted into the anterior chamber parallel to the iris and the aqueous humor in the anterior chamber is aspirated until the anterior chamber has completely collapsed. Then the eyeball is split in half with a sterile scalpel and the gelatinous vitreous is removed with sterile cutlery (tweezers, sharp spoons, etc.). Finally, if the retina does not detach you from the rest of the procedure, carefully separate it from the rest of the eye and remove it with tweezers.
Alle so gewonnenen Augenanteile werden zur weiteren Bearbeitung in separate sterile Probengefäße verbracht . Zur Gewinnung einer Probe vom lebenden Tier wird z.B. ein Rind zunächst sediert . Alternativ kann das Tier auch in Vollnarkose gelegt werden, wenn es sich um ein nicht zur Lebensmittelgewinnung genutztes Tier bzw. um ein sehr scheues (Zoo-) Tier handelt. Unter Anwendung allgemein üblicher Zwangsmaßnahmen (Fußfesseln, Kopfstrick) wird ein Lokal- anästhetikum auf die Kornea des zu untersuchenden Auges auf- getropft und der restliche Kopf abgedeckt. Anschließend wird mit einem sterilen In ektionsbesteck am temporalen Rand der Kornea am Übergang zur Sklera eingestochen und die Injektionsnadel parallel zum Verlauf der Iris in die vordere Augenkammer eingebracht und Kammerwasser aspiriert .All eye parts obtained in this way are placed in separate sterile sample vessels for further processing. For example, a cattle is first sedated to obtain a sample from the living animal. Alternatively, the animal can also be put under general anesthesia if it is an animal not used for food production or a very shy (zoo) animal. Using general coercive measures (ankle cuffs, head rope), a local anesthetic is dripped onto the cornea of the eye to be examined and the rest of the head is covered. Subsequently, a sterile induction set is inserted at the temporal edge of the cornea at the transition to the sclera, the injection needle is inserted into the anterior chamber of the eye parallel to the course of the iris and aspirated aqueous humor.
Die vom lebenden oder toten Tier entnommenen Augenanteile werden zur Herstellung der Probe nachfolgend mechanisch oder physikalisch zerkleinert und damit homogenisiert. Die Zerkleinerung kann z.B. mittels Ultraschall oder eines schnellaufenden Rührwerks, z.B. einem Ultrathurrax, erfolgen. Die hergestellte homogenisierte Flüssigkeit kann nachfolgend fil- tiert oder zentrifugiert werden. Die Probe kann dann aus dem Überstand gewonnen werden.The parts of the eye removed from the living or dead animal are subsequently mechanically or physically comminuted and thus homogenized to produce the sample. The comminution can e.g. by means of ultrasound or a high-speed agitator, e.g. an Ultrathurrax. The homogenized liquid produced can subsequently be filtered or centrifuged. The sample can then be obtained from the supernatant.
Die Probe kann mittels herkömmlicher Verfahren unmittelbar auf das Vorliegen von PrPsc untersucht werden. Das nach dem Stand der Technik derzeit verwendete langwierige Aufbereitungsverfahren einer aus dem Gehirn entnommenen Probe entfällt. The sample can be examined directly for the presence of PrP sc using conventional methods. The lengthy preparation process currently used according to the prior art of a sample taken from the brain is eliminated.
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| DE10108099.9 | 2001-02-19 | ||
| DE2001108099 DE10108099A1 (en) | 2001-02-19 | 2001-02-19 | Method for detection of specific proteins and use |
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| EP1437144A1 (en) * | 2003-01-09 | 2004-07-14 | IVPT Gmbh | Rapid in-vivo test for Transmissible Spongiform Encephalopathy in the eye |
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| US4615877A (en) * | 1984-02-27 | 1986-10-07 | Savapa S.R.L. | Method of determining the oxidizing activity of a biological liquid, and a corresponding reagent |
| JP3333213B2 (en) * | 1996-04-03 | 2002-10-15 | スティヒティング インスティチュート フォール ディールハウデレイ エン ディールゲゾントヘイト | How to detect prion disease |
| US5985581A (en) * | 1996-07-25 | 1999-11-16 | The Mclean Hospital Corporation | Use of presenilin-1 for diagnosis of alzheimers disease |
| DE19741607A1 (en) * | 1997-09-20 | 1999-03-25 | Prionics Ag | New polypeptides comprising prion protein sequences |
| US6165784A (en) * | 1997-10-14 | 2000-12-26 | The United States Of America As Represented By The Secretary Of Agriculture | Antibodies for the detection of prion protein as an indication of transmissible spongiform encephalopathies |
| DE19918141A1 (en) * | 1999-04-21 | 2000-10-26 | Boehringer Ingelheim Vetmed | Diagnosing transmissible spongiform encephalopathy, particularly before appearance of clinical symptoms, by detecting specific markers in blood cells |
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| EP1437144A1 (en) * | 2003-01-09 | 2004-07-14 | IVPT Gmbh | Rapid in-vivo test for Transmissible Spongiform Encephalopathy in the eye |
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| WO2002066987A3 (en) | 2003-01-09 |
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