WO1991018276A1 - Dispositif de microdosage a bandelettes pour dosage immunologique - Google Patents

Dispositif de microdosage a bandelettes pour dosage immunologique Download PDF

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Publication number
WO1991018276A1
WO1991018276A1 PCT/US1991/003589 US9103589W WO9118276A1 WO 1991018276 A1 WO1991018276 A1 WO 1991018276A1 US 9103589 W US9103589 W US 9103589W WO 9118276 A1 WO9118276 A1 WO 9118276A1
Authority
WO
WIPO (PCT)
Prior art keywords
immunochemical
immunochemical reagent
labelled
microassay
capillary
Prior art date
Application number
PCT/US1991/003589
Other languages
English (en)
Inventor
Kenneth A. Kelly
Benjamin D. Card
Nevio Recinelli
Simonetta Cosimi
Original Assignee
Kelly Kenneth A
Card Benjamin D
Nevio Recinelli
Simonetta Cosimi
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 Kelly Kenneth A, Card Benjamin D, Nevio Recinelli, Simonetta Cosimi filed Critical Kelly Kenneth A
Publication of WO1991018276A1 publication Critical patent/WO1991018276A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N35/1065—Multiple transfer devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00178—Special arrangements of analysers
    • G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00465—Separating and mixing arrangements
    • G01N2035/00564—Handling or washing solid phase elements, e.g. beads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/026—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having blocks or racks of reaction cells or cuvettes

Definitions

  • Analytes such as antibodies or antigens
  • biological fluids such as serum
  • the reagent, antibody or antigen, which reacts specifically with the analyte is labelled to permit measurement and quantitation of the analyte.
  • the labels may be radioactive emitters such as Iodine-125, or enzymes such as urease or alkaline phosphatase which on reaction with substrate produce a color or fluorescence change which can be measured optically.
  • Solid phase immunoassays utilize a solid support, such as paper discs, plastic test tubes, cellulose threads, icrotiter plastic wells, to which one of the reactants in the immunoassay is attached. Solid phase immunoassays facilitate the separation of the bound phase of the reaction, which is the immune complex formed on the surface of the solid phases, from the free phase of the reaction which is the reagent(s) in the surrounding fluid.
  • Patent 3,867,517 Catt, K.J., 1972, U.S. Patent 3,646,346).
  • Coated tubes are bulky reagents from a commercial production point of view and do not lend tne selves readily to automation.
  • the most significant improvement in automated solid phase immunoassay is the use of microtiter wells, made of polystyrene or polyvinyl chloride and usually contain 0.5 milliliters of reagent. These wells can be moulded into strips of 8 to 12 wells or plates of 8 x 12 wells (Voller, A., Bidwell, D.E., Huldt, G. and Bartlett, A., 1974, Bull. WHO, 5_1, 209-221) .
  • Instrumentation has been developed for automation of immunoassays carried out in microtiter wells to dispense reagents into the wells, to wash the wells to remove free reactants and to measure the end product of the immunoassay reaction, such as a color change in substrate reagent in the well.
  • a capillary device for multiple analytes has been developed by K.A. Kelly (PCT Patent Application WO 89/00290) in which polyvinyl chloride solid rod-like segments, 10mm long by 1.8mm diameter, are coated with allergens and inserted in a capillary tube of 2.3mm inner diameter.
  • a syringe is used to fill and empty the capillary device of reagents. This device has been applied to the detection of IgE antibodies to multiple allergens in an enzyme immunoassay.
  • the present invention describes a device, the "ENEAs" microassay strip, which can be used to carry out a solid phase immunoassay for analytes in a fluid.
  • the device consists of a rectangular strip containing a plurality of vertical U-shaped reaction wells of capillary dimensions. In the larger capillary chamber of each reaction well is inserted a rod-like segment, to which surface has been attached a reagent which reacts specifically with the analyte in the fluid.
  • the strip may contain twenty-five of these capillary wells.
  • the strip is designed for use in an automated enzyme immunoassay: The analyte fluid is dispensed into the chamber containing the reactive segment and allowed to react forming an insolubilized immune complex on the segment surface.
  • the free analyte in solution is removed by addition of wash fluid into the reaction arm of the U-chamber and aspiration from the other arm of smaller diameter.
  • substrate is added.
  • the amount of analyte on the surface of the segment is estimated by measurement of the color change in the reaction chamber spectrophotonietrical ly through the wall of the chamber.
  • Figure 1 is a sectional view through the microassay strip device 21 showing:
  • Figure 2 is a schematic representation of a coordinate work station with microassay strips in place in an automated immunoassay.
  • FIG. 3 is an enlarged portion of the microassay strip device showing the U-shaped chamber 23 and reactive segment 25.
  • FIG. 1 An example of the "EHEAs" microassay strip 21 in accordance with the present invention is illustrated in Fig. 1.
  • the strip is basically a rectangular piece with a length of 145mm, height 16mm and thickness 3mm. It contains twenty-five U-shaped reaction chambers 23; however, the actual number of chambers may vary.
  • Each U-shaped chamber (Fig. 3) consists of one vertical arm 23A of inner diameter of
  • the above dimensions of the microassay strip and U-shaped chambers are preferred dimensions though not essential to the present invention.
  • the microassay strip may be made from transparent or optically clear glass or plastic material such as polystyrene or polyvinyl chloride or other optically clear polymeric materials.
  • the capillary U-shaped reaction chamber as described permits reagent fluid to be added to or removed from either arm of the chamber.
  • the larger bore chamber is designed to contain one rod-like plastic segment 25, 10mm height by 1.8mm diameter, with an immunoassay reagent affixed to the surface of the segment.
  • the volume of reagent required to fill the U-shaped reaction chamber as described is less than 50 microtiters.
  • the "ENEAs" microassay strip in accordance with the present invention has wide application to the detection of analytes in a fluid, it is especially well suited for solid phase immunoassays.
  • the "ENEAs" microassay strip is used for the detection of antibodies of the IgE class in the serum or plasma of allergic patients by a conventional enzyme immunoassay.
  • Allergenic reagents such as aqueous extracts from grass, weed or tree pollens, from animal dander or from various food substances are attached to the surface of the rod-like segments.
  • Exemplary materials of the solid phase segments include glass or plastic polymers such as polystyrene, polyvinyl chloride, etc.
  • the allergenic reagents may to attached to the solid phase surface by known techniques, such as passive adsorption or covalent bonding.
  • Exemplary procedures of covalent boding involve treating the polymer surface with glutaraldehyde prior to reaction with the allergen in a 2-step procedure, or reacting mixture of allergen and glutaraldehyde with the polymer surface in a 1-step procedure (Parsons, G.H., 1978, U.S.
  • Patent 4,069,352 The allergen solid phase is subsequently treated with an inert substance, such as albumin, to saturate residual active sites on the surface and to stabilize the coated surface for long term use.
  • the allergen coated segment is next placed in the larger bore chamber for immunoassay.
  • fluid serum sample (40-50ul) 27 is dispensed into the chamber.
  • the reaction is allowed to proceed for a sufficient time at a suitable temperature to allow first antibodies in the serum to complex specifically with allergens on the segment surface. Typical conditions of incubation are 60 minutes at ambient room temperature (18-30C).
  • the chamber and segment within are washed repeatedly to remove free fluid reactants .
  • a suitable enzyme-second antibody reagent such as Urease coupled covalently to antibody specific for human IgE, is next ad ⁇ e and incubated under appropriate conditions to allow the enzyme-second antibody to form immune complexes with the IgE first antibody on the segment surface.
  • the chamber and segment within are again washed repeatedly to remove free enzyme-antibody reactants.
  • a substrate- indicator system for Urease enzyme such as a urea-bromocreso1purp1e pH indicator solution mixture, is next added to the chamber.
  • Urease enzyme such as a urea-bromocreso1purp1e pH indicator solution mixture
  • a positive yellow to purple color change will occur which can be measured by placing the microassay strip in a suitable spectrophotometer.
  • One modification of the "ENEAs" microassay strip 21 involves attaching the first immunochemical reagent of antibody or antigen directly to the inner wall of the U-shaped capillary chamber, rather than to a reactive segment which can be placed within the capillary chamber.
  • Microassay strips prepared by this modification may be prepared with attached immunochemical reagents as described for reactive segments and may be used in an immunoassay in a similar manner to microassay strips containing reactive segments within the U-shaped capillary chambers.
  • the "ENEAs" microassay strip may be used for a variety of solid phase immunoassays wherein one of the antibody or antigen reactants i attached to the surface of the solid phase to insolubilize by immune co plexing with the other reactant in the fluid phase.
  • the labels in the detection system may include any combination of enzyme/substrate systems, or antibody or antigen reactants labelled with fluorescent markers such as fluorescein, with appropriate optical instrumentation for measurement.
  • the "ENEAs" microassay strip is particularly suited for an automated immunoassay system.
  • an automated system depicted generally in Fig. 2 a large number of these immunoassay strips are fixed on a movable platform.
  • the system can be programmed to perform a multitude of steps in sequence. These steps include addition of a number of samples of unknown analyte concentrations, standards and controls samples to appropriate reaction U-chambers, washing after a predetermined incubation time, addition of labelled second reactant, washing after a predetermined incubation time, followed by measurement and printout of results.
  • the advantage of this system is that it is fully automated after loading of reagents and programming.
  • the unique features of a capillary U-shaped reaction chamber are that fluid reagent volumes are minimal (_ ⁇ 50ul ) , there is a large surface area to volume ratio which maximizes the rate of immunochemical reactions to equilibrium, and proper washing of the solid phase after each reaction step can be carried out more easily and thoroughly.
  • Wash solution reagent is dispensed into one arm of the U-shaped chamber 23 and removed by suction applied at the other arm of the chamber. This provides a unidirectional flow through the chamber which more efficiently removes fluid reactants in the chamber. Instrumentation to dispense and aspirate wash solution for the U-shaped chamber are available and may be easily incorporated in the automated system (Fig. 2).
  • Strips can be prepackaged in which all the solid phase segments per strip are coated with the same allergen reactant.
  • a number of analyte serum samples and standards may be added to the strip to quantitate the level of antibody in the samples to that particular allergen.
  • Another application involves assembling segments coated with difference allergens in the single microassay strip and adding a single analyte sample to each well of the strip to quantitate IgE antibodies to a panel of allergens.
  • a third application involves the use of segments coated with antibody to human IgE in the strip so that samples can be assayed for their level of IgE immunoglobulin.
  • reactive segments 25 may be inserted into the U-shaped chambers 23, stabilizing liquid added if necessary, and the upper surface of the microassay strip 21 sealed of the U-chambers until use in assay.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Clinical Laboratory Science (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

Dispositif de microdosage à bandelettes à chambres multiples (21) destiné à la détection d'analytes dans des liquides biologiques par dosage immunologique. Les chambres (23) des bandelettes sont en forme de U et peuvent être recouvertes de réactifs immunochimiques ou peuvent contenir des segments en phase solide (25) recouverts de réactifs immunochimiques. Les bandelettes de microdosage (21) sont particulièrement destinées à être utilisées dans un système de dosage immunologique automatique.
PCT/US1991/003589 1990-05-22 1991-05-21 Dispositif de microdosage a bandelettes pour dosage immunologique WO1991018276A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US52706990A 1990-05-22 1990-05-22
US527,069 1990-05-22

Publications (1)

Publication Number Publication Date
WO1991018276A1 true WO1991018276A1 (fr) 1991-11-28

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE9203583U1 (de) * 1992-03-17 1992-05-07 Alcan Deutschland GmbH, 3400 Göttingen Näpfchenplatte
EP0518827A2 (fr) * 1991-06-13 1992-12-16 Simonetta Cosimi Système (réactif et instrument) pour determiner de facon complètement automatique la présence d'immunoglobulines E totales ou dirigée contre allergènes, ou d'autres molécules dans des échantillons aqueux
US6165798A (en) * 1996-10-10 2000-12-26 University Of British Columbia Optical quantification of analytes in membranes
US7097974B1 (en) 1998-08-28 2006-08-29 Febit Biotech Gmbh Support for a method for determining an analyte and a method for producing the support
US7470540B2 (en) 2000-10-17 2008-12-30 Febit Ag Method and device for the integrated synthesis and analysis of analytes on a support
ITCS20120019A1 (it) * 2012-04-20 2013-10-21 Uni Degli Studi Del Molise Dispositivo, metodo e kit per il rilevamento di differenti marker in differenti tipi cellulari o molecolari e loro quantificazione

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4146365A (en) * 1977-11-07 1979-03-27 Litton Bionetics, Inc. Affinity detection apparatus
EP0154687A2 (fr) * 1982-03-03 1985-09-18 Becton Dickinson and Company Plaque de support et dispositif d'examen pour les essais immunologiques
EP0174247A2 (fr) * 1984-08-23 1986-03-12 Bernard Guerin Bandelette d'analyse immunologique et procédé pour sa fabrication
EP0197729A2 (fr) * 1985-04-01 1986-10-15 Donald Patrick Kelly Procédé d'essai immunologique enzymatique
US4756884A (en) * 1985-08-05 1988-07-12 Biotrack, Inc. Capillary flow device
US4849340A (en) * 1987-04-03 1989-07-18 Cardiovascular Diagnostics, Inc. Reaction system element and method for performing prothrombin time assay

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4146365A (en) * 1977-11-07 1979-03-27 Litton Bionetics, Inc. Affinity detection apparatus
EP0154687A2 (fr) * 1982-03-03 1985-09-18 Becton Dickinson and Company Plaque de support et dispositif d'examen pour les essais immunologiques
EP0174247A2 (fr) * 1984-08-23 1986-03-12 Bernard Guerin Bandelette d'analyse immunologique et procédé pour sa fabrication
EP0197729A2 (fr) * 1985-04-01 1986-10-15 Donald Patrick Kelly Procédé d'essai immunologique enzymatique
US4756884A (en) * 1985-08-05 1988-07-12 Biotrack, Inc. Capillary flow device
US4849340A (en) * 1987-04-03 1989-07-18 Cardiovascular Diagnostics, Inc. Reaction system element and method for performing prothrombin time assay

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0518827A2 (fr) * 1991-06-13 1992-12-16 Simonetta Cosimi Système (réactif et instrument) pour determiner de facon complètement automatique la présence d'immunoglobulines E totales ou dirigée contre allergènes, ou d'autres molécules dans des échantillons aqueux
EP0518827A3 (en) * 1991-06-13 1993-01-20 Simonetta Cosimi System (reagent and instrument) for the automatic determination of the presence of total or allergen specific immunoglobulin e, or other molecules in aqueous samples
DE9203583U1 (de) * 1992-03-17 1992-05-07 Alcan Deutschland GmbH, 3400 Göttingen Näpfchenplatte
US6165798A (en) * 1996-10-10 2000-12-26 University Of British Columbia Optical quantification of analytes in membranes
US7097974B1 (en) 1998-08-28 2006-08-29 Febit Biotech Gmbh Support for a method for determining an analyte and a method for producing the support
EP1742058A2 (fr) 1998-08-28 2007-01-10 Febit Biotech GmbH Substrats pour procédés de détermination d'analytes et méthodes de fabrication de tels substrats
US7737088B1 (en) 1998-08-28 2010-06-15 Febit Holding Gmbh Method and device for producing biochemical reaction supporting materials
US7470540B2 (en) 2000-10-17 2008-12-30 Febit Ag Method and device for the integrated synthesis and analysis of analytes on a support
ITCS20120019A1 (it) * 2012-04-20 2013-10-21 Uni Degli Studi Del Molise Dispositivo, metodo e kit per il rilevamento di differenti marker in differenti tipi cellulari o molecolari e loro quantificazione

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