EP0888534A1 - Systeme d'evaluation quantitative a resolution locale d'elements d'analyse - Google Patents

Systeme d'evaluation quantitative a resolution locale d'elements d'analyse

Info

Publication number
EP0888534A1
EP0888534A1 EP97916377A EP97916377A EP0888534A1 EP 0888534 A1 EP0888534 A1 EP 0888534A1 EP 97916377 A EP97916377 A EP 97916377A EP 97916377 A EP97916377 A EP 97916377A EP 0888534 A1 EP0888534 A1 EP 0888534A1
Authority
EP
European Patent Office
Prior art keywords
zone
analyte
test element
detection zone
detection
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Withdrawn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
EP97916377A
Other languages
German (de)
English (en)
Inventor
Peter Blasberg
Franz Bolduan
Susanne Gentsch
Hans-Peter Haar
Erich Händler
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.)
Roche Diagnostics GmbH
Original Assignee
Roche Diagnostics GmbH
Boehringer Mannheim GmbH
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 Roche Diagnostics GmbH, Boehringer Mannheim GmbH filed Critical Roche Diagnostics GmbH
Publication of EP0888534A1 publication Critical patent/EP0888534A1/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Withdrawn legal-status Critical Current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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G01N21/8483—Investigating reagent band
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex

Definitions

  • the present invention relates to a system for the quantitative, spatially resolved detection of an analyte using a test element, including
  • test element with a sample application zone which contains a labeled substance which binds specifically to the analyte and a detection zone which contains an area which serves to immobilize a complex of analyte and labeled substance
  • a plurality of sensors are arranged in a row and this linear array is moved transversely to the row of sensors by a transport device so that an area is covered,
  • the present invention is in the area of the quantitative evaluation of test elements by transmission or reflection measurement
  • the European patent application EP-A-0 646 784 describes a video system for evaluating test elements, in which test elements are placed on a support by the user. The test elements are evaluated without prior positioning and the video system used must therefore be Capture the entire pad to ensure that the detection zone is in the image field. This system ensures a very high degree of user-friendliness, since the user does not have to carry out any positioning, but this results in a greatly reduced accuracy of the analysis result, since only relatively few sensors of the video system are available for evaluating the actual test field. In order to grant the user the desired freedom when storing a test element, the apparatus described must also have a large opening area through which stray light can penetrate. The apparatus described is therefore only suitable for quantitative evaluations in which the signal to be measured is large and the accuracy requirements are low
  • DE 3247355 Devices for evaluating thin-layer chromatograms are described in documents EP-B-0 437 968 and DE 3247355.
  • the device of DE 3247355 is a scanner with which a thin-layer chromatography plate is scanned.
  • the flat elements viewed by a single sensor have a size of about 0.05 to 0.2 mm.
  • the object of the present invention was to improve the existing equipment for evaluating test elements to such an extent that clinically relevant analytes which occur in very low concentrations (pico-mol) can be quantitatively determined with high accuracy. It was a further object of the invention to find a device which is easy to use and inexpensive for this purpose
  • Test elements which can be used for the present invention are referred to in the prior art as immunological test elements.
  • Test elements suitable for the invention have one or more absorbent matrices on a carrier material, "the matrices being arranged on the carrier material in such a way that a Liquid flow through all matrices is possible.
  • a suitable test element has a sample application and a detection zone. Furthermore, it can also have a zone for separating interfering substances, for example erythrocytes.
  • the carrier material of the test element should make it possible to fix the matrices on it and make the entire arrangement manageable. Carrier materials should therefore be sufficiently rigid and chemically inert for the examination to be carried out.
  • plastic films for example made of polysterol, have proven to be suitable
  • the one or more matrices can be attached to the carrier material, for example by double-sided adhesive tape or with a hot melt adhesive.
  • the matrices are arranged on the carrier material in such a way that they form a liquid transport path and liquid, which is applied to the sample application zone, reaches the detection zone by capillary forces .
  • a so-called suction zone to the detection zone, which consists of a material that absorbs liquids well.
  • specifically bindable substance there is a labeled substance in the sample application zone that binds specifically to the analyte. If, for example, saccharides are to be detected, the corresponding lectins can be used as a specifically binding substance. In the detection of DNA or RNA, complementary DNA or RNA strands can be considered as the specifically bindable substance.
  • a particularly important class of specifically bindable substances are antibodies or antibody fragments which bind to the corresponding analytes, which are antigens. Further specifically bindable substances are known in the prior art
  • the specifically bindable substance has a label.
  • Chromophore or fluorophoric groups can be considered here due to the optical evaluation in reflection or transmission.
  • Corresponding dyes are sufficiently known in the prior art.
  • Gold marking has been found to be particularly suitable for the application according to the invention.
  • the term "marking" is also intended to include groups which are not themselves colored, but by a chemical reaction or by specific binding to a colored molecule can be detected.
  • porous, fibrous or non-fibrous materials have proven to be the most suitable matrix material Proven Suitable " Papers, nonwovens, porous plastic layers and membranes may be mentioned here.
  • Fibrous matrices which contain a high proportion of glass fibers and / or synthetic fibers, such as polyester fibers and / or cellulose, have proven particularly suitable During the liquid transport, a complex is formed from the analyte to be detected and the labeled substance, which also migrates with the liquid stream.This complex arrives with the liquid stream into the detection zone.
  • the detection zone is the zone that is illuminated and is detected by the reflected or transmitted radiation Detection zone must therefore be made that it is optically as homogeneous as possible so as not to interfere with the detection process. Any inhomogeneities can, however, be taken into account to a certain extent in the evaluation, provided that they are the same before and after the detection reaction. However, such optical inhomogeneities that only occur when the detection zone is moistened should be avoided
  • the detection zone has an area which serves to immobilize a complex of analyte and labeled substance. This area can be generated by applying a substance that binds the complex to the detection zone. It has proven to be advantageous that the substance that binds the complex transversely to the flow direction to be applied as a line or thin tape Lines of several tens of millimeters to several millimeters, preferably of 0.5 to 1 mm in width, have proven to be very suitable for the detection of analytes in concentrations of approximately 10 12 mol / l which bind the complex Substance in turn must be bound to the matrix material so that it enables the complex to be immobilized.
  • the substance binding the complex can be applied, for example, with a cannula by an inkjet process or an airbrush in the desired width. It has proven advantageous proven if the detection zone in this area consists of cells There is urozitrate or nitrocellulose ester, since very many substances, in particular proteins or nucleic acids, are strongly absorptively bound to it. It is thus possible to bind the specific binding substance sufficiently firmly to the matrix by pure absorption. Of course, it is also possible that covalently bind the complex immobilizing substance to the matrix
  • test elements that can be used in the context of the invention can be found in EP-A-0 323 605 and EP-A-0 186 799. These literature references refer in particular to matrix materials, chromogenic or fluorophoric groups , full content "Referring specifically binding substances, substances for immobilization of complexes of analyte and labeled substance and the production of these test elements
  • the amount of labeled substance in excess of the amount of analyte is advantageously selected so that quantitative binding of the analyte to the labeled substance
  • the substance that is used to immobilize the complex of analyte and labeled substance should bind to the labeled substance as little as possible in order to avoid falsification of the analysis result.
  • the immobilization zone passes, the complex is intercepted from the liquid, while unbound labeled substance moves on.
  • the amount of analyte or amount of sample that is applied to the sample application zone should be large enough to transport the unbound labeled substance out of the area of the detection zone, so that a falsification of the analysis result by labeled substance that is not bound to analyte is avoided If the amount of sample is not sufficient, an auxiliary liquid, such as water, isotonic saline or detergent solution, can be added to the sample application zone
  • the use of the type of test element mentioned in the context of the present invention has the advantage that the amount of analyte and thus also the amount of complex of analyte and labeled substance is concentrated in a relatively small and well-defined range Analyte concentration reaches a high signal level, which has a more favorable signal-to-noise ratio than would be the case if the label to be detected were distributed over a larger area, as is the case, for example, with thin-layer chromatograms. Furthermore, the use of a specifically bindable Substance for labeling the analyte and also the specific immobilization of the complex formed the advantage that the analyte is separated from other substances
  • a test element according to the invention is advantageously located in a housing in which it is held.
  • the housing has an opening for feeding the sample liquid onto the sample application zone and an opening through which the detection zone is optically accessible.
  • the housing generally consists of a base and a base Upper part, between which the test element is clamped By struts and the like, it is ensured that the test element is in a well-defined position within the housing.
  • the housing serves to mechanically stabilize the test element, to enable easier positioning and the test element to be made accessible through openings at the necessary points.
  • materials are used for the housing that have sufficient rigidity " In particular, plastics such as polyethylene or polystyrene are possible here.
  • An apparatus is used to evaluate a test element which has reacted with test liquid, which enables a spatially resolved evaluation.
  • This apparatus contains a holder for the test element or a housing, in which a test element is located, for positioning the detection zone.
  • An insert can be used as a holder, for example, in which the test element is pushed onto stop edges, through which it is positioned within a plane.
  • the test element is preferably also pressed onto the base from the top in order to position it vertically
  • a system according to the invention also has an illumination device for illuminating the detection zone of the test element.
  • the lighting device can have a broadband spectrum.
  • an illumination device with a narrow-band spectrum is preferred.
  • light-emitting diodes have proven to be advantageous.
  • halogen lamps in combination with spectral filters are also suitable
  • the lighting device is installed so that the detection zone is illuminated as homogeneously as possible.
  • this can be achieved with a circular arrangement of light-emitting diodes in a plane parallel to the plane of the detection zone, the radiation cone of the light-emitting diodes being directed at the plane of the detection zone. It is not necessary to focus the light-emitting diode on the detection zone.
  • the frequency band of the lighting device such that it falls within the absorption range of the marking of the marked substance.
  • green light-emitting diodes have been well preserved as light sources.
  • blue light-emitting diodes are particularly suitable. It is known in the prior art to control the light sources in such a way that their radiation intensity is as constant as possible during the recording of the measured values, for example by pulsing the light sources with a suitable current. The measurement is carried out during a plateau phase of the light pulse
  • the detection zone is illuminated by the lighting device and detected by it reflected or transmitted by a sensor array.
  • a sensor array is either a two-dimensional array, for example a CCD array, or a linear array that is moved across the row of sensors with a transport device, so that an area is covered.
  • Two-dimensional sensor arrays are already relatively inexpensive due to their use in video cameras available Arrays suitable for the use according to the invention should be at least approximately Have 250,000 individual sensors. With a size of the detection zone in the range of 1 cm x 1 cm, a resolution of approximately 0.1 mm in each spatial direction is achieved. Increasing the resolution has an advantageous effect on the evaluation accuracy
  • a linear sensor array is used, either a classic transport device can be used with which the sensor array is displaced transversely to the row of sensors, but it is also possible to optically image individual strips of the detection zone on the line array using an optical arrangement.
  • An apparatus according to DE 3247355 is suitable for this, for example.
  • a device according to P 19520606.1 can also be used particularly advantageously.However, two-dimensional sensor arrays offer the advantage that no moving parts are required in the apparatus. However, one-dimensional arrays are much cheaper and easier to control if several thousand pixels per row are to be resolved
  • An essential aspect of the present invention is that the spatially resolved evaluation of the detection zone and in particular the area around the immobilization zone is carried out with a sufficient number of sensors. In addition to a sufficient number of individual sensors, this requires a suitable optical imaging of the detection zone on the sensor array. The optical imaging should take place in such a way that the image of the detection zone fills the area of the sensor array or the area covered by a linear array as completely as possible.
  • An arrangement according to the invention has the advantage over a quantitative evaluation of thin-layer chromatograms that the area in which the detectable signal occurs is predetermined by the immobilization zone. It is therefore possible to adjust the size of the detection zone, the size of the sensor array and the optical imaging means to one another that the sensor array is used optimally.
  • this is achieved by means of a weak reduction optics for imaging the detection zone on the sensor array.
  • an image converter which consists of a large number of optical fibers, all of which have a cross section at one end that corresponds in size and shape to the detection zone, the other end in size and shape corresponds to the sensor array.
  • image converters are available, for example, from Schott or Galileo Electro-Optics Corporation
  • the illumination devices are preferably arranged around the edge of the imaging system.
  • LEDs can be grouped in a circle around the lens.
  • Such an arrangement of the light emitting diodes improves the homogeneity optical illumination A number of 4 to 30 light-emitting diodes has proven to be well suited
  • the system according to the invention has a converter which digitizes these signals.
  • Such converters are sufficiently known in the prior art and are generally referred to as analog-digital converters.
  • the digitized sensor signals are generally obtained from an evaluation device a micro computer, evaluated
  • test elements according to the invention there is a distribution of the detection signal which is essentially determined by the shape of the immobilization zone.
  • the concentration of the marking substance is high in the area facing the application zone, while it decreases in the direction of the suction matrix. It has been found that For the exact quantitative evaluation of this type of test element, an integral measurement is not sufficient. On the other hand, it has been shown that with the spatially resolved evaluation according to the invention, a very precise quantitative evaluation is possible.
  • each individual sensor signal is converted into a quantity of substance and based on it the total amount of substance determined in the individual measurement. With knowledge of the amount of sample applied to the test element, the concentration of analyte can be determined.
  • the advantage of the spatially resolved measurement is that the non-linear relationship between concentration and remission or absorption can be better taken into account.
  • the accuracy of the evaluation depends crucially on how strong the concentration within the surface area, which is detected by a single sensor of the sensor array, is constant.
  • the shape of the immobilization zone, the distribution curve of the marking substance is largely predetermined. It is therefore possible to adapt a spline function on the basis of the measured values, so that interpolation between the individual measured values is possible when calculating the amount of immobilized marking substance.
  • This increases the evaluation accuracy in particular when the signals from two adjacent sensors are relatively far apart , since this indicates that the concentration or amount of marking substance fluctuates relatively strongly within the area that is detected by a single sensor
  • the signals averaged over the columns in turn represent a line which reproduces the signal curve perpendicular to the detection lines.
  • light materials such as papers or nonwovens
  • the signal curve along the analytical test element which is determined by a reflectance measurement, therefore has minima at the locations of the detection zones.
  • a spatially resolved evaluation of test elements also offers the advantage that a correction of the background is possible.
  • the background which is caused by a coloration of the detection zone that is not caused by the substance to be detected, can only be in Form of a constant are taken into account.
  • the spatially resolved method of the present invention it is possible to appropriately limit the area that is being evaluated based on the signal distribution, so that background effects outside the selected area have no influence on the measurement result.
  • the measured signal curve is analyzed and signals from Sensors that lie below or above a determined threshold value are not taken into account in later calculations.
  • the slope of the signal curve can also be used as a criterion for limiting the evaluation range, or a maximum distance from the minima of the remission curve is specified in order to cover the evaluation range limit Due to the already mentioned signal curve, which can be approximated by a function curve, it is also possible with this function curve by extrapolation to also detect areas in which the sensor signals are due to the threshold value correction were not taken into account
  • the evaluation device turns the sensor signals that are evaluated or from values that are determined by a functional curve into a quantity of substance This is usually done by comparing the sensor signals with signals that have been determined for known concentrations of analyte.
  • the creation of calibration curves which allow an assignment of reflectance or transmission values to the respective amount of substance or concentration is well known in the prior art.
  • Figure 3 cross section through a housing with test element
  • FIG. 1 shows an analysis element in which a sample application zone (1), an erytrocyte separation zone (2), a detection zone (3) and a suction zone (4) are applied to a carrier material (5).
  • a sample application matrix (6) is arranged in the sample application zone (1), which partially overlaps an erytrocyte separation matrix (7).
  • the erytrocyte separation matrix (7) in turn overlaps the detection matrix (8) (detection zone) on which an immobilized substance is in the form a line (9) is applied.
  • a suction matrix (10) slightly overlaps the detection matrix (8). All of the reagents that are required to form a complex with the analyte to be detected are accommodated in the sample application matrix (6).
  • the test element presented here as an example serves to determine the concentration of troponin T from whole blood.
  • Troponin T is an important indicator for the determination of cardiac muscle necrosis.
  • the sample application zone consists of two superposed nonwovens, the first being impregnated with a gold-labeled antibody against Troponin T and the second nonwoven containing a biotinylated antibody against Troponin T.
  • a line (9) of streptavidin is applied within the detection zone
  • Figure 2 shows a plan view of a housing (12) with a test element.
  • the housing has two openings, the sample application opening (13) and the detection opening (14) Both openings taper in the direction of the test element.
  • the housing also has a holding part (15) to improve handling
  • Figure 3 shows a cross section through a housing with test element
  • the test element lies on the lower housing part (17), while the upper housing part (16) with the housing support (18) rests on the stops (19) of the lower housing part (17), so that a defined Ab ⁇ the lower and upper housing parts can be guaranteed.
  • the test element is secured against slipping by one stop (19) and another stop (20), so that a defined position of the sample application zone and the detection zone relative to the openings in the housing is ensured can be
  • FIG. 3 also shows the application of a sample liquid (23) with the aid of a pipette (22) to the sample application zone.
  • the sample releases biotinylated antibodies and gold-labeled antibodies from the matrices (11 and 26), which bind with the analyte of the sample liquid
  • biotinylated antibodies and gold-labeled antibodies from the matrices (11 and 26), which bind with the analyte of the sample liquid
  • Different complexes between analyte and the two named antibodies, which are in equilibrium with one another, are particularly important for the detection of complexes that have both biotinylated antibodies and gold-labeled antibodies.
  • the sample liquid with the complexes migrates through the erytrocyte separation matrix (7 ) and arrives in the detection zone (8). Due to the streptavidin strip (9) applied to the detection zone, biotinylated antibodies are immobilized. Only complexes which contain both biotin and gold markings contribute to the detectable signal.
  • FIG. 4 shows a holder into which a housing with a test element is inserted.
  • the holder has a flat base plate (30) on which two stop edges (31, 32) are mounted.
  • FIG. 5 shows a cross section through the system according to the invention.
  • the detection zone (8) and in particular the immobilization line (9) are imaged onto the sensor array (41) by an optical system (40), which is shown schematically as a lens.
  • the illumination of the detection zone takes place via light-emitting diodes (42) which are arranged around the sensor array (41).
  • the microprocessor (CPU) controls the sensor array.
  • Analog signals of the sensor array are converted into digital signals with an A / D converter (A / D), which are fed to the microprocessor (CPU)
  • the sensor signals are also evaluated in the microprocessor in order to determine the concentration of analyte.
  • the analysis result is shown on a display (D)
  • FIG. 6 shows a signal curve of a test element with a control line.
  • the figure shows the signal of one line of a two-dimensional sensor array with 165 lines and 192 columns.
  • the respective column of the sensor array is indicated on the X axis of the display.
  • the Y axis shows the gray value of the respective sensors in arbitrary units.
  • the detection line at clinically relevant concentrations of troponin T has a relatively small signal swing compared to the control line. This illustrates the need for a spatially resolved measurement for this area of application.
  • edge effects are noticeable, which occur due to shadows caused by the housing. This shows that it is of crucial importance to use a spatially resolved evaluation method with this type of test element which can correct or eliminate these effects.

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

Abstract

L'invention concerne un système d'évaluation quantitative à résolution locale d'éléments d'analyse, comprenant un support pour un élément d'analyse, grâce auquel l'élément d'analyse peut être positionné, un élément d'analyse muni d'une zone de dépôt de l'échantillon à analyser et d'une zone de détection, un dispositif d'éclairage, une barrette de détecteurs, un convertisseur, une unité d'évaluation servant à évaluer des signaux numériques, ainsi qu'un système de visualisation pour afficher les résultats de l'évaluation.
EP97916377A 1996-03-22 1997-03-22 Systeme d'evaluation quantitative a resolution locale d'elements d'analyse Withdrawn EP0888534A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19611347 1996-03-22
DE1996111347 DE19611347A1 (de) 1996-03-22 1996-03-22 System zur quantitativen ortsaufgelösten Auswertung von Testelementen
PCT/EP1997/001469 WO1997036168A1 (fr) 1996-03-22 1997-03-22 Systeme d'evaluation quantitative a resolution locale d'elements d'analyse

Publications (1)

Publication Number Publication Date
EP0888534A1 true EP0888534A1 (fr) 1999-01-07

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EP97916377A Withdrawn EP0888534A1 (fr) 1996-03-22 1997-03-22 Systeme d'evaluation quantitative a resolution locale d'elements d'analyse

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Country Link
EP (1) EP0888534A1 (fr)
JP (1) JP2000507353A (fr)
DE (1) DE19611347A1 (fr)
WO (1) WO1997036168A1 (fr)

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Publication number Publication date
JP2000507353A (ja) 2000-06-13
WO1997036168A1 (fr) 1997-10-02
DE19611347A1 (de) 1997-09-25

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