EP1430309A2 - Bande de test permettant de deceler des proteines prions - Google Patents

Bande de test permettant de deceler des proteines prions

Info

Publication number
EP1430309A2
EP1430309A2 EP02781187A EP02781187A EP1430309A2 EP 1430309 A2 EP1430309 A2 EP 1430309A2 EP 02781187 A EP02781187 A EP 02781187A EP 02781187 A EP02781187 A EP 02781187A EP 1430309 A2 EP1430309 A2 EP 1430309A2
Authority
EP
European Patent Office
Prior art keywords
test strip
sample
detection
test
test strips
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02781187A
Other languages
German (de)
English (en)
Inventor
Paul Price
Bruno Oesch
Eric KÜBLER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Prionics AG
Original Assignee
Prionics AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Prionics AG filed Critical Prionics AG
Publication of EP1430309A2 publication Critical patent/EP1430309A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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 test strip according to the preamble of claim 1 and a holder with which several test strips can be used simultaneously in different sample vessels.
  • Test strips are used for the detection of a number of different analytes in liquid or homogenized samples.
  • at least one delimited area is provided on the test strip, in which a detection reagent for the specific analyte is immobilized.
  • test strips are brought into contact with the sample with a lower section.
  • the delimited detection area is either in the lower section of the test strip and is then wetted directly by the sample.
  • a further possibility is to provide the detection area further above on the test strip, in which case, however, the strip is made of a liquid-conducting material and the sample liquid can flow from the contact section to the detection area due to capillary action. Whether a proof is positive or negative can e.g. B. can be observed by discoloration in the detection area.
  • test strips are mainly used for such evidence that is particularly easy to standardize or that can be carried out by people who are otherwise not knowledgeable at home etc.
  • Known examples are e.g. B. pregnancy tests or test strips to check the kidney function.
  • the object of the present invention is therefore to provide a device with which prions in liquid or liquefied or homogenized samples can be detected much more easily than hitherto.
  • Another object of the invention is to provide a device with which the simultaneous measurement of several samples can be carried out in a particularly simple manner. The problems are solved with a test strip that has the characterizing features of claim 1 and with a device that has the characterizing features of claim 9.
  • the test strip according to the invention has a lower section which can be brought into contact with a liquid or homogenized sample, a first delimited area being provided according to the invention, in which antibodies or other detection reagents are immobilized, which form the prion protein tie.
  • Suitable antibodies are known to the person skilled in the art from the publication of "Korth, C. et al; Nature 1997, Vol 390, pages 74-77".
  • test strip is made according to claim 1 from an absorbent material, in particular nitrocellulose or polyvinylidene fluoride (PVDF), so that a liquid transport between the lower portion that can be brought into contact with the sample and the first delimited area is possible.
  • an absorbent material in particular nitrocellulose or polyvinylidene fluoride (PVDF)
  • PVDF polyvinylidene fluoride
  • the sample is first mixed with a detection reagent such as antibodies which, together with a marker, form a detectable complex with the prion protein.
  • a detection reagent such as antibodies which, together with a marker, form a detectable complex with the prion protein.
  • Markers such as radioactive isotopes, fluorescent substances, light-absorbing chromophores etc. in the UV or visible range can be used for the detection.
  • markers can be, for example, colored polymer balls such as latex beads, gold particles, liposomes and dye particles or the like.
  • all detectable markers that bind or are bound to antibodies or other detection reagents actively or passively can. For the sake of simplicity, only examples with antibodies bound to colored markers are described below.
  • test strip is brought into contact with the sample and the sample liquid moves over the first delimited area in which the detection reagents immobilized there bind the prion protein contained in the sample with the colored marker attached to it.
  • the first delimited area There is discoloration in the first delimited area, e.g. can be observed with the eye.
  • Suitable detection reagents are e.g. Antibodies, aptamers or other specific recognition organs for prion proteins.
  • one or more further delimited areas can be provided on the test strip, in which control reagents are immobilized.
  • the test strip in this context has a second delimited area in which reagents are immobilized which can bind the colored reagent-marker complex in the sample.
  • the control reagents used for this can be, in particular, an antibody or a functional reagent that only specifically bind the colored reagent-marker complex. if it can still bind explicitly to the prion protein.
  • recombinant prion protein can be used as the control reagent.
  • a further advantageous embodiment relates to test strips which are used in detection reactions for PrP Sc . It is known that prion proteins are present in two different isoforms, called PrP c and PrP Sc . PrP c is the isoform of a normal mammalian protein, while PrP Sc is an abnormal, pathological isoform.
  • PrP Sc is specific for prion diseases. Usual tests and detection methods are based on PrP Sc as a disease marker and in particular check the presence of this molecule in samples.
  • a third delimited area can be used in the test strip according to the invention be provided in which control reagents are immobilized which specifically recognize and bind the N-terminus of the PrP protein. With complete digestion, the N-terminal region of PrP should no longer be detectable. If, on the other hand, there is discoloration in this third area, this indicates that PrP c is still intact in the sample and that the digestion was therefore not complete.
  • Suitable control reagents can be, for example, the antibodies known from the publication by "Barry, RA et al .; J. Immunol. 1988, Vol. 140, pages 1188-1193".
  • an area designated as a waste pad can also be provided, which absorbs the liquid that has passed through the strip.
  • an absorbent mat, or a fleece or blotting paper or the like may be provided.
  • the shape and depth of the test strip can be designed in such a way that a small chromatography column is created which allows PrP to be separated and detected even from larger volumes.
  • test strips according to the invention with waste pad described above are preferably made of absorbent material so that the liquid from the sample can flow over the possibly different detection and control areas.
  • test strips that are not made of absorbent material.
  • the delimited detection and possibly provided control areas would have to be provided on the test strip in such a way that they can be wetted directly with the sample or the liquid has to be moved actively (eg by suction or centrifugation).
  • the invention relates not only to the test strips mentioned, but also to a device for testing several samples simultaneously.
  • the preferred device according to the invention accordingly has a holder in which a plurality of test strips are arranged and received in such a way that their lower sections can be inserted simultaneously into one of the sample vessels of the composite used.
  • a further embodiment of the invention provides a holder which at the same time holds all the test strips required for a specific form.
  • a holder for a microtiter plate in which one of the sample vessels (cavities) number corresponding number of test strips is received.
  • such a device could have, for example, a frame in which a strip-shaped holder with a corresponding number of test strips can be inserted for each row of the microtiter plate. To read the results, the holders can be removed from the frame one after the other, which simplifies the evaluation.
  • FIG. 2 shows an embodiment of a holder in which a plurality of test strips are fastened in a row parallel to one another;
  • 3 shows a frame in the usual microtiter plate format, into which the frame shown in FIG.
  • FIG. 4 shows a frame fully equipped with holders according to FIG. 2
  • Fig. 3; Fig. 5 shows the results of an investigation in which several in one as in
  • Fig. 2 shown holder attached test strips for the detection of recombinant bovine prion protein different
  • FIG. 6 shows the results of an investigation with several test strips
  • Wild types 7 shows the results of an investigation with two test strips for the detection of disease-specific, protease-resistant prion protein
  • test strip 10 for the detection of prion proteins.
  • the test strip has a lower section 11 which can be brought into contact with a homogenized or liquid sample.
  • test strip 10 contains detection or control reagents.
  • detection or control reagents can e.g. B. applied by spraying on the test strip 10.
  • the test strip 10 consists of absorbent material, e.g. B. nitrocellulose. Sample liquid which comes into contact with the test strip 10 in the section 11 is sucked through the test strip along the regions 14, 12, 13 up to a waste pad 15 which absorbs the liquid which has passed through. An identification 16 is provided at the upper end of the test strip. B. indicates the coordinates of the sample in a microtiter plate.
  • each test strip contains reagents that may recognize prion protein contained in the sample.
  • these reagents which are special antibodies against the prion protein, are contained in the delimited region 12.
  • the area 13 contains control reagents with which the concentration or the presence of the colored detection reagent-marker complex mentioned above in the sample can be checked.
  • the area 14 finally contains reagents with which the digestion of prion proteins can be checked.
  • FIG. 2 shows a device 20 with which several samples can be analyzed simultaneously.
  • the device 20 has a holder 21, on which test strips 10, 10 ′, etc. are accommodated in parallel alignment with their downward-pointing sections 11.
  • the distance between the strips 10, 10 'to each other is chosen so that it z. B. corresponds to the usual distance between cavities in a microtiter plate.
  • the holder 21 can then be used to simultaneously check samples in the cavities of a row of a microtiter plate.
  • Perforations 22 can be formed between the individual test strips 10, 10 'on the holder 21, which in the case shown is in the form of a strip, which make it easier to separate individual test strips 10, 10'.
  • a frame 30 is shown in FIG. 3, the base area of which is selected such that it can be placed on a conventional microtiter plate with an adapter 32 and then completely covers it.
  • Opposite pairs of slots 31, 31 'etc. are provided in the area of the upper longitudinal edges 30, into each of which a holder 21 with the test strips 10, 10' can be inserted.
  • the number of opposing pairs of slits 31, 31 ' corresponds to the number of rows in a microtiter plate, so that a holder 21 can be used for each row of a microtiter plate.
  • FIG. 4 shows the fully inserted state of such a holder 30, the adapter 32 being omitted in this illustration.
  • test strips shown each contain a delimited area 12, 13, 14 which contain the different detection and control reagents.
  • the invention is also intended to cover embodiments in which not only one but several delimited areas are provided on the test strip per reagent, e.g. two or more delimited areas, each carrying detection reagents that recognize prion protein contained in the sample, or two or more areas that contain digestion reagents, etc.
  • FIG. 5 shows the results of an investigation with several test strips for the detection of recombinant bovine prion protein (RecBoPrP) of different concentrations.
  • the test strips were incubated with RecBoPrP of various starting dilutions using the methods outlined above. The dilution of the samples ranges between 1: 1 and 1:32. The respective dilutions are indicated under the test strips on the figure.
  • a test strip was incubated with a sample that did not contain RecBoPrP.
  • test strip While area 13 of the test strip, which reacts to the presence of the detection reagent-marker complex in the sample, is constantly colored, area 12 of the test strip, which recognizes PrP 27-30, has a decreasing value in accordance with the decreasing concentration of RecBoPrP Dye intensities. Within the 32-fold concentration range of the RecBoPrP one can see clearly distinguishable coloring intensities of the area 12 of the test strips.
  • the test strips can therefore also be used for the semi-quantitative detection of RecBoPrP.
  • FIG. 6 shows three rows 60, 60 'and 60 ", each with several test strips after incubation with urine from four transgenic mice or wild-type mice, respectively.
  • test strips in turn have the area 12 which recognizes PrP 27-30 and the area 13 which reacts to the presence of the detection reagent-marker complex in the sample.
  • the series 60 test strips were incubated with urine from four wild-type mice (WT1-WT4) producing normal amounts of prion protein, resulting in a clear staining of area 12 in the test strips.
  • the 60 'series test strips were incubated with urine from four transgenic mice (Tg20 1- Tg20 4) which produce greatly increased amounts of prion protein. This leads to an even clearer coloring of area 12 in the test strips.
  • test strips were incubated with urine from four transgenic mice (Prnp% l-Prnp% 4) which do not produce prion protein.
  • the area 12 in the test strips is therefore not colored.
  • FIG. 7 shows test strips A and B with which brain homogenate of a healthy cow and a cow suffering from BSE treated with protease were examined.
  • PrP c the non-infectious isoform of the prion protein
  • PrP Sc infectious isoform
  • test strips A and B each again have the area 12 which recognizes PrP 27-30, and the area 13 which reacts to the presence of the detection reagent-marker complex in the sample.
  • Test strip A was incubated with protease-treated brain homogenate from a healthy cow. The area 12 of the test strip remains undyed because the PrP c has been digested completely.
  • Test strip B was incubated with protease-treated brain homogenate from a cow suffering from BSE. It can be clearly seen that the area 12 of the test strip is colored. Despite the protease treatment, PrP 27-30 is therefore present, which suggests that the sample contained PrP Sc before digestion.
  • a strip test of the type shown here is therefore suitable for rapid BSE screening of bovine brain samples.
  • 8 shows test strips A, B and C which were incubated with different homogenates which were digested to different degrees.
  • test strips A, B, C have, in addition to regions 12 that recognize PrP 27-30, and 13 that react to the presence of the detection reagent-marker complex in the sample, regions 14 that are the N-terminal region of the prion protein bind and thus only recognize undigested or incompletely digested prion protein, but not undigested prion protein, which lacks the N-terminal region.
  • Test strip A was incubated with completely digested protease-treated brain homogenate from a cow suffering from BSE. Area 12 is colored because the homogenate contains PrP27-30, while area 14 remains uncolored because there are no more N-terminal areas due to complete digestion.
  • Test strip B was incubated with fully digested protease-treated brain homogenate from a healthy cow. This sample contains neither PrP 27-30 nor N-terminal areas, so that areas 12 and 14 remain unstained.
  • Test strip C was incubated with incompletely digested protease-treated brain homogenate from a healthy cow.
  • the area 12 is colored because the homogenate still contains the domain PrP 27-30 also contained in PrP c because of the poor digestion, while the area 14 is colored because N-terminal areas are also still present. Without region 14 it would not have been possible to differentiate with certainty whether the coloration of region 12 gives an indication of PrP 27-30 from a positive sample or is due to insufficient digestion of normal prion protein.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

L'invention concerne une bande de test permettant de déceler une substance à analyser contenue dans des échantillons liquides ou homogénéisés. Cette bande comprend une section pouvant être mise en contact avec l'échantillon et au moins une première zone délimitée sur la bande de test. Dans cette première zone délimitée, sont immobilisés des réactifs de détection qui lient la protéine prion. L'invention concerne également un système permettant de tester simultanément plusieurs échantillons dans des récipients d'échantillons qui sont regroupés dans une disposition géométrique définie. Ce système présente un support dans lequel plusieurs bandes de test sont maintenues dans une disposition correspondant à la disposition géométrique définie des récipients d'échantillons de telle façon que sa section inférieure puisse simultanément être introduire dans respectivement un récipient d'échantillon.
EP02781187A 2001-09-25 2002-09-24 Bande de test permettant de deceler des proteines prions Withdrawn EP1430309A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10147012 2001-09-25
DE10147012 2001-09-25
PCT/EP2002/010681 WO2003029813A2 (fr) 2001-09-25 2002-09-24 Bande de test permettant de deceler des proteines prions

Publications (1)

Publication Number Publication Date
EP1430309A2 true EP1430309A2 (fr) 2004-06-23

Family

ID=7700082

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02781187A Withdrawn EP1430309A2 (fr) 2001-09-25 2002-09-24 Bande de test permettant de deceler des proteines prions

Country Status (7)

Country Link
US (1) US20040241877A1 (fr)
EP (1) EP1430309A2 (fr)
JP (1) JP2005504320A (fr)
AU (1) AU2002349311B2 (fr)
CA (1) CA2453589A1 (fr)
NZ (1) NZ530626A (fr)
WO (1) WO2003029813A2 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004008539A1 (de) * 2004-02-19 2005-09-01 Prionics Ag Vorrichtung und Verfahren zur optischen Auswertung von Teststreifen
EP1596199A1 (fr) * 2004-05-14 2005-11-16 Prionics AG Méthode de détection des formes pathogènes des protéines prions.
JP4903001B2 (ja) * 2006-02-24 2012-03-21 クレトイシ株式会社 レジノイド砥石の製造方法
JP5917951B2 (ja) * 2012-03-02 2016-05-18 日研ザイル株式会社 抗酸化能判定具
US20150017066A1 (en) * 2013-07-09 2015-01-15 IMMCO Diagnostics, Inc. Line immunoassay testing device
US10119964B2 (en) * 2014-12-22 2018-11-06 Digital Biotech, LLC Multiplex assay strip, beads, device and method
JP6889990B2 (ja) * 2016-07-29 2021-06-18 株式会社カネカ 検査具
CN111492241B (zh) 2017-09-29 2023-04-18 由联邦材料研究和检测机构主席所代表的经济与能源部长所代表的德意志联邦共和国 检测土壤和水中的烃污染
WO2022035726A1 (fr) * 2020-08-10 2022-02-17 Envirologix Inc. Systèmes et procédés pour une utilisation de multiples bâtonnets diagnostique

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DE3382430D1 (de) * 1982-03-03 1991-11-14 Becton Dickinson Co Trageplatte und anordnung mit mehreren bechern fuer immunologische untersuchungen.
DE3416933A1 (de) * 1984-05-04 1985-11-07 Dora 1000 Berlin Köhler Mit antigenen oder antikoerpern beschichteter traeger
US4891321A (en) * 1987-10-21 1990-01-02 Hubscher Thomas T Apparatus for performing determinations of immune reactants in biological fluids
US5451504A (en) * 1991-07-29 1995-09-19 Serex, Inc. Method and device for detecting the presence of analyte in a sample
AU4925993A (en) * 1992-09-18 1994-04-12 Abbott Laboratories Multiple assay test strip devices
SE9203320D0 (sv) * 1992-11-06 1992-11-06 Pharmacia Lkb Biotech A method of processing nucleic acid samples
WO1998022824A1 (fr) * 1996-11-18 1998-05-28 Avraham Zer Bandes d'essai d'immuno-chromatographie
AU738606B2 (en) * 1997-02-06 2001-09-20 Enfer Technology Limited Immunological assay for spongiform encephalopathies
EP0861900A1 (fr) * 1997-02-21 1998-09-02 Erziehungsdirektion Of The Canton Zurich Détection immunologique de prions
EP0973034A1 (fr) * 1998-07-16 2000-01-19 Microbe Scope AG Immunoessais et dispositifs pour leur fonctionnement
FI982481A0 (fi) * 1998-11-17 1998-11-17 Wallac Oy Immunomääritys nautaeläinten tarttuvan spongiomuotoisen aivotaudin määrittämiseksi
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Also Published As

Publication number Publication date
AU2002349311B2 (en) 2007-11-08
WO2003029813A3 (fr) 2004-02-26
WO2003029813A2 (fr) 2003-04-10
JP2005504320A (ja) 2005-02-10
NZ530626A (en) 2007-11-30
US20040241877A1 (en) 2004-12-02
CA2453589A1 (fr) 2003-04-10

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