WO1991002073A1 - Enceinte a vide pour immunoanalyse possedant une plaque microtitre - Google Patents

Enceinte a vide pour immunoanalyse possedant une plaque microtitre Download PDF

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Publication number
WO1991002073A1
WO1991002073A1 PCT/US1990/004086 US9004086W WO9102073A1 WO 1991002073 A1 WO1991002073 A1 WO 1991002073A1 US 9004086 W US9004086 W US 9004086W WO 9102073 A1 WO9102073 A1 WO 9102073A1
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WO
WIPO (PCT)
Prior art keywords
holes
plate
chamber
membrane
solution
Prior art date
Application number
PCT/US1990/004086
Other languages
English (en)
Inventor
Carl R. Clark
Original Assignee
Pierce Chemical Company
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 Pierce Chemical Company filed Critical Pierce Chemical Company
Publication of WO1991002073A1 publication Critical patent/WO1991002073A1/fr

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Classifications

    • 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
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B01L3/50255—Multi-well filtration
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

  • the present invention relates to immunoassay procedures and, in particular, to quantitative immunoassay procedures which can be rapidly accomplished in substantially less time than heretofore obtainable.
  • Immunoassay procedures have for many years provided sensitive diagnostic tools for the detection of a variety of substances, generally referred to as ligands. Such procedures are described in a number of articles and texts, an example of which is Reviews on Immunoassav Technology, Ed. S. B. Pal, Pub. Chapman & Hall, 1988.
  • ELISA immunoassay procedure
  • a solid support such as the well in a plastic plate in accomplishing the assay.
  • a receptor for a target ligand is bound to the solid support.
  • a liquid sample containing the ligand, having specificity for the bound receptor is then applied to the plate.
  • an enzyme conjugate having binding specificity for the ligand is added to the well.
  • a substrate is added which develops color on contact with the bound enzyme, the amount of ⁇ olor developed being dependent upon the amount of bound conjugate present which, in turn, is indicative of the amount of target ligand bound to the support.
  • concentration of ligand in the sample can be determined.
  • conventional ELISA procedures take on the order of five hours or more.
  • a nitrocellulose filter is pre-coated with an antigen. Thereafter, a solution containing the target ligand, in this case an antibody, is drawn through the filter followed by rinsing solutions.
  • an enzyme-labeled antibody (Ijsselmuiden (1987)) or 125 I-labeled protein A (Ijsselmuiden (1989)), both of which have binding specificity for the target ligand, is applied and drawn through the filter to detect the target antibody bound to the antigen on the nitrocellulose filter.
  • the device used to accomplish the above described assay is illustrated in the 1989 Ijsselmuiden article. It consists of three blocks of perspex which are clamped together during the assay.
  • the bottom section has an external outlet and a valve, and constitutes a reservoir attached to the upper sections.
  • the middle and top sections, designed to accommodate the nitrocellulose filter between them, contain 32 corresponding holes with a diameter of 5 mm and neoprene "O" rings facing the nitrocellulose sheet to prevent lateral flow. While Ijsselmuiden appears to describe an immunoassay procedure which has the advantage of rapidity over prior procedures, only the procedure utilizing 125 I detection is quantitative.
  • the enzyme- labeled antibody system (Ijsselmuiden (1987)) yields only a qualitative determination of target ligand.
  • a procedure having the foregoing attributes which also can utilize commercially available microtiter plates and associated readers for determination of color development would be advantageous.
  • the present invention embodies the features of filtration, such as illustrated by Ijsselmuiden. But, in addition thereto, it provides means for collecting the colored reaction product of enzyme and substrate in a fashion that permits quantitative measurement of color development in a microtiter plate system to achieve quantitation of target ligand.
  • the present invention provides an apparatus which can be used for colorimetric ligand- receptor assay procedures to quantitatively determine the concentration of a target ligand in a liquid sample.
  • the apparatus contains a top member, a middle member and a bottom member in a sandwiched relationship.
  • the top and middle members are plates having holes therethrough and between which a membrane can be placed.
  • the holes are in axial alignment and the cross-sectional area of the holes at the bottom surface of the top plate is greater than the cross-sectional area of the holes at the top surface of the middle plate.
  • the sidewalls of the holes in " the middle plate extend below the surface of the plate.
  • the holes in the middle plate are preferably contained within tubes, herein termed cannulas, which are inserted through openings initially formed in the middle plate.
  • the bottom member of the apparatus is a collection chamber which has an opening on the upper side which faces the bottom surface of the middle plate.
  • the chamber contains a port through one of its surfaces so that a vacuum can be created within the chamber.
  • the chamber contains means for accepting a microtiter plate containing a plurality of wells. In the assembled apparatus, the ends of the sidewalls of the holes which extend beneath the bottom surface of the middle plate are located within the wells of the microtiter plate.
  • the apparatus contains means for securing the three members together in a vacuum type relationship when a membrane is positioned between the top and middle plates.
  • the apparatus described above is first assembled ith a liquid permeable membrane, to which a receptor can be bound, placed between the top and middle plates, and without the plate containing the wells being positioned in the bottom chamber.
  • the membrane can either have the receptor already bound thereto or a solution containing the receptor can be added to the holes in the top plate.
  • vacuum is then created in the chamber with, for example, a peristaltic pump, so that the solution can be pulled past the membrane at a highly controlled consistent rate.
  • the liquid sample containing the target ligand is added to the holes in the top plate.
  • vacuum is applied and the liquid is drawn directly through the membrane, through the holes in the middle plate, and then discharged into the- collection chamber.
  • the ligand is bound to the receptor on the membrane.
  • a solution containing an enzyme conjugate which has binding specificity for the target ligand is drawn through the membrane and is, in turn, bound to the target ligand. This step is then followed by a washing step to remove enzyme' conjugate which did not bind to the ligand.
  • the bottom chamber is separated from the top and middle plates, which remain secured together, and is emptied of any liquid which may be present from previous steps. Then, the microtiter plate is positioned in the bottom chamber and the apparatus is reassembled with the sidewall extensions of the middle plate now extending into the wells of the microtiter plate.
  • Sufficient substrate solution is utilized so that on collection in the wells the solution extends above the bottom ends of the sidewall extensions. This feature is important in order to insure that the volume of solution transferred from the holes in the upper plate to the wells in the microtiter plate is the same for each set of holes and wells in the apparatus. In turn, this permits measurement of well to well differences in color intensity which correlate to differences in bound ligand.
  • the bottom chamber is again separated from the remaining structure, the microtiter plate removed from the chamber and the development of color of the solution in the wells determined.
  • FIG. 1 is an exploded perspective view showing the apparatus of the present invention with certain parts omitted and with a membrane included.
  • FIG. 2 is an exploded side elevation with certain parts broken away and shown in section.
  • FIG. 3 is a cross-section taken vertically, through the apparatus.
  • FIG. 4 is an enlargement, partly in section, of one of the clamps.
  • FIG. 5 is a top plan view of the assembled apparatus.
  • FIG. 6 is a side elevation, of view of the assembled apparatus.
  • the apparatus illustrated in the drawings is shown to comprise as a top member a sample application plate 10 having a plurality of holes 12 extending therethrough into which a liquid sample can be placed.
  • the middle member is a membrane support plate 14 positioned beneath the plate 10 with the liquid permeable membrane 16, when present, being placed between the two plates.
  • the membrane support plate 14 has openings 18 into which the cannulas 20 are inserted.
  • each cannula has a sidewall portion 22 with a hole 24 extending through the cannula.
  • the holes 12 in the application plate 10 are in axial alignment with the holes 24 in the cannulas 20 located in the support plate 14.
  • the support plate contains studs 26 which are adapted to fit into the holes 30 of the application plate.
  • Four thumb screws 34 serve to secure the application plate 10 and membrane support plate 14 together.
  • the cannulas 20 contain a sidewall portion 22 and a hole 24.
  • the sidewalls 22 of the cannulas extend beneath the bottom surface of the support plate 14.
  • the sidewalls of the cannulas extend above the top surface of the middle plate, the top end of the cannulas containing a flanged portion 36 which abuts against the membrane 16.
  • the holes are conically shaped and, as illustrated, the cross-sectional area of the holes 12 at the bottom surface of the plate 10 is greater than the cross-sectional area of the holes 24 at the top surface of the cannulas 20.
  • the cross- sectional area of the flanged portion 36 of the cannulas 20 is greater than the cross-sectional area of the holes 12 at the bottom surface of the application plate.
  • flexible "O" rings 38 are placed below the flanged portion 36 of the cannulas between the flanged portions and the membrane support plate 14.
  • other gasketing means can be used to prevent lateral dispersion of liquid through the membrane.
  • a flexible perforated diaphragm having dimensions co-extensive with the array of holes in the top plate, which can be disposed on either or both sides of the flanged portions of the cannulas, a diaphragm on both sides being preferred.
  • the illustrated apparatus is shown to contain, as a bottom member, a collection chamber 40 for receiving liquid drawn through the application plate 10, the membrane 16 and the holes 24 in the support plate 14.
  • the collection chamber is open on the upper side thereof which faces the bottom surface of the membrane support plate.
  • the chamber contains the vacuum port 42 which is attached via tubing and valving to a vacuum pump such as a peristaltic pump.
  • the bottom internal surface 44 of the collection chamber 40 is sloped to permit fluid to flow to the vacuum port and, in turn, be removed from the chamber 40.
  • the chamber 40 also contains a port 46 which contains a valve (not shown) to relieve vacuum in the chamber after each sample solution (i.e., receptor, ligand, etc.) has been drawn through the membrane.
  • sample solution i.e., receptor, ligand, etc.
  • the final step in the assay procedure described herein involves collecting the colored liquid reaction product of substrate and enzyme so that color can be measured and, in turn, the amount of bound target ligand determined.
  • a microtiter plate 50 containing a plurality of wells 52 for collection of liquid.
  • the lower portion of the microtiter plate contains a skirt 54 which, when the plate is inserted into the chamber 40, rests upon the ledge 56 in the chamber.
  • the ledge 56 is, as indicated, discontinuous between points 58 and 60.
  • the end of the sidewall extension of the cannulas 20 is located within the wells 52 of the microtiter plate 50.
  • the extent to which the end of the cannulas so extend into the wells is such that when the assay procedure is completed and the colored reaction product of substrate and enzyme has been drawn into the wells 52, the end of the cannulas are submerged below the surface of the liquid.
  • the collection chamber is secured in vacuum tight relationship to the membrane support plate 14 by means of conventional clamping and gasketing.
  • An example of such is shown in the drawings to include clamps 62 and gasket 48.
  • the clamps 62 they are shown in FIG. 4 to include a lever 64, a bracket 66 which is secured to the collection chamber 40 by eans of the screws 68, a bracket 70 which is secured to .the membrane support plate by means of the screws 72,. and a*tension screw 74 adjustably mounted in the housing 76.
  • the housing 76 is pivotally mounted at the base of the lever 64 which, in turn, is pivotally mounted on the bracket 66.
  • the upper bracket 70 is slotted to accommodate the head of the screw 74.
  • the head of the screw 74 is inserted ijito the slot of the bracket 70 by pivoting the screw into position. Then, the lever 64 is raised, thus forcing the screw and, in turn, the bracket 70 downward to, in combination with the gasket 48, to effectively seal the chamber to the membrane support plate.
  • the tension can be adjusted to insure a vacuum seal.
  • the following example illustrates the use of the above described apparatus. All parts of the apparatus, except for the thumb screws and clamps, were made of plastic and a peristaltic pump was used to create vacuum.
  • a commercially available nitrocellulose membrane sold for use in immunoassays was employed. Prior to insertion between the sample application plate and the support plate, the membrane was wetted with distilled water. The apparatus when assembled measured about 6-1/2" x 4-1/2" and was approximately 2-1/2" high. 96 conically shaped holes were present in the application plate.
  • the membrane support plates contained 96 cannulas as illustrated.
  • a commercially available microtiter plate with 96 wells was used. The procedure employed was as - follows.
  • HSA human serum albumin
  • the vacuum relief valve was then opened and again closed after which time 200 ⁇ l of a 3% bovine serum albumin (BSA) solution was added to each of the holes in the application plate. Over a five minute period, the BSA solution was drawn through the membrane and discharged into the collection chamber, this step serving to block the vacant binding sites on the membrane. Again, the vacuum relief valve was opened and closed. Next, 200 ⁇ l of a 1:1000 dilution (in Tris buffer) of mouse derived anti-HSA was deposited into each of the holes in the application plate and drawn through the membrane and discharged into the chamber over a five minute period.
  • BSA bovine serum albumin
  • the vacuum relief valve was opened and the collection chamber separated from the top assembly of the apparatus containing the application plate, membrane and membrane support plate.
  • the chamber was emptied of any liquid remaining from the foregoing operations and the top assembly was placed on a piece of paper toweling to remove any excess liquid off the bottom of the cannulas.
  • a commercially available 96 well flat bottomed microtiter plate was placed in the collection chamber and the apparatus reassembled by clamping the collection chamber and the membrane support plate together. As so assembled, the bottom of the cannulas extend into the wells of the microtiter plate.
  • the protocol illustrated as an example above is commonly called a direct sandwich assay
  • the apparatus and methodology herein described is not limited to this type of assay. It can also be used in direct assays, indirect assays, indirect sandwich assays, competitive assays, etc. Essentially any immunoassay arrangement used with standard ELISA type techniques can be mimicked with the apparatus and methodology described herein.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
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  • Food Science & Technology (AREA)
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Abstract

On décrit un appareil d'immunoanalyse qui sert à la détermination quantitative de la concentration d'un ligand cible dans un échantillon liquide. L'appareil comprend des plaques supérieure (10) et intermédiaire (14) possédant des trous (12, 18). Ces trous sont axialement alignés et les trous dans la plaque intermédiaire possèdent des parois latérales (22) qui s'étendent en-dessous de la surface inférieure de la plaque intermédiaire. Une membrane (16) perméable aux liquides est située entre les plaques. L'appareil comprend une enceinte de fond (40), qui est ouverte à la surface inférieure de ladite plaque intermédiaire, et un orifice extracteur d'air (42), et peut contenir une plaque microtitre qui comprend une pluralité de réservoirs (52). Du liquide est placé dans les trous dans la plaque supérieure et l'on crée un vide dans l'enceinte. Le liquide est entraîné à vitesse réglée directement à travers la membrane sans qu'il n'y ait de dispersion latérale, à travers les trous dans la plaque intermédiaire et jusqu'à l'enceinte de fond.
PCT/US1990/004086 1989-07-28 1990-07-20 Enceinte a vide pour immunoanalyse possedant une plaque microtitre WO1991002073A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US38622689A 1989-07-28 1989-07-28
US386,226 1989-07-28

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CA (1) CA2063986A1 (fr)
WO (1) WO1991002073A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0502371A2 (fr) * 1991-03-07 1992-09-09 Eppendorf-Netheler-Hinz Gmbh Dispositif d'aspiration pour plaques de microtitration à membranes
WO1999019067A1 (fr) * 1997-10-10 1999-04-22 Biosepra, Inc. Pile multicoupelles a plusieurs plaques alignees et procede de traitement d'echantillons biologiques/chimiques de ladite pile
WO2001020325A1 (fr) * 1999-09-16 2001-03-22 Immunetics, Inc. Dosages immunologiques de membrane permettant de detecter plusieurs maladies transmises par des tiques
WO2009112952A2 (fr) * 2008-03-12 2009-09-17 Cellectricon Ab Appareil et procédé destinés à l’alignement de pointes de plaques à plusieurs puits
US8057754B2 (en) 2008-03-12 2011-11-15 Cellectricon Ab Apparatus and method for tip alignment in multiwell plates
WO2012120169A1 (fr) 2011-03-09 2012-09-13 Zf Biotox, S.L. Microplaque pour essais biologiques

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4031197A (en) * 1973-04-25 1977-06-21 Gte New Ventures Corporation In vitro method for determining allergic hypersensitivity
US4427415A (en) * 1979-01-05 1984-01-24 Cleveland Patrick H Manifold vacuum biochemical test method and device
WO1985003886A1 (fr) * 1984-02-29 1985-09-12 Gerhard Noss Procede et installation pour amener simultanement une pluralite d'echantillons de liquides sur une lame porte-objets
US4599315A (en) * 1983-09-13 1986-07-08 University Of California Regents Microdroplet test apparatus
EP0197729A2 (fr) * 1985-04-01 1986-10-15 Donald Patrick Kelly Procédé d'essai immunologique enzymatique
US4626509A (en) * 1983-07-11 1986-12-02 Data Packaging Corp. Culture media transfer assembly
EP0233385A1 (fr) * 1986-01-13 1987-08-26 De Staat Der Nederlanden Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Procédé de détection d'un membre d'une paire ligand-récepteur, méthode de préparation d'un support auquel le membre est lié et équipement d'analyse à cet effet
US4761378A (en) * 1983-03-04 1988-08-02 American Home Products Corp. (Del.) Microbiological testing apparatus
US4834946A (en) * 1987-02-05 1989-05-30 Levin Andrew E Apparatus for blot screening numerous, small volume, antibody solutions
US4895706A (en) * 1986-10-28 1990-01-23 Costar Corporation Multi-well filter strip and composite assemblies

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4031197A (en) * 1973-04-25 1977-06-21 Gte New Ventures Corporation In vitro method for determining allergic hypersensitivity
US4427415A (en) * 1979-01-05 1984-01-24 Cleveland Patrick H Manifold vacuum biochemical test method and device
US4761378A (en) * 1983-03-04 1988-08-02 American Home Products Corp. (Del.) Microbiological testing apparatus
US4626509A (en) * 1983-07-11 1986-12-02 Data Packaging Corp. Culture media transfer assembly
US4599315A (en) * 1983-09-13 1986-07-08 University Of California Regents Microdroplet test apparatus
WO1985003886A1 (fr) * 1984-02-29 1985-09-12 Gerhard Noss Procede et installation pour amener simultanement une pluralite d'echantillons de liquides sur une lame porte-objets
EP0197729A2 (fr) * 1985-04-01 1986-10-15 Donald Patrick Kelly Procédé d'essai immunologique enzymatique
EP0233385A1 (fr) * 1986-01-13 1987-08-26 De Staat Der Nederlanden Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Procédé de détection d'un membre d'une paire ligand-récepteur, méthode de préparation d'un support auquel le membre est lié et équipement d'analyse à cet effet
US4895706A (en) * 1986-10-28 1990-01-23 Costar Corporation Multi-well filter strip and composite assemblies
US4834946A (en) * 1987-02-05 1989-05-30 Levin Andrew E Apparatus for blot screening numerous, small volume, antibody solutions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF IMMUNOLOGICAL METHODS, Volume 119, 1989, IJESSELMUIDEN et al., "Optimizing the Solid-Phase Immunofiltration Assay", pp. 35-43. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0502371A2 (fr) * 1991-03-07 1992-09-09 Eppendorf-Netheler-Hinz Gmbh Dispositif d'aspiration pour plaques de microtitration à membranes
EP0502371A3 (en) * 1991-03-07 1993-01-20 Eppendorf-Netheler-Hinz Gmbh Suction device for membrane microtiter plates
WO1999019067A1 (fr) * 1997-10-10 1999-04-22 Biosepra, Inc. Pile multicoupelles a plusieurs plaques alignees et procede de traitement d'echantillons biologiques/chimiques de ladite pile
US6464942B2 (en) 1997-10-10 2002-10-15 Ciphergen Biosystems, Inc. Plate alignment and sample transfer indicia for a multiwell multiplate stack and method for processing biological/chemical samples using the same
US7273759B2 (en) 1997-10-10 2007-09-25 Pall Corporation Plate alignment and sample transfer indicia for a multiwell multiplate stack and method for processing biological/chemical samples using the same
WO2001020325A1 (fr) * 1999-09-16 2001-03-22 Immunetics, Inc. Dosages immunologiques de membrane permettant de detecter plusieurs maladies transmises par des tiques
WO2009112952A2 (fr) * 2008-03-12 2009-09-17 Cellectricon Ab Appareil et procédé destinés à l’alignement de pointes de plaques à plusieurs puits
WO2009112952A3 (fr) * 2008-03-12 2010-04-08 Cellectricon Ab Appareil et procédé destinés à l’alignement de pointes de plaques à plusieurs puits
US8057754B2 (en) 2008-03-12 2011-11-15 Cellectricon Ab Apparatus and method for tip alignment in multiwell plates
WO2012120169A1 (fr) 2011-03-09 2012-09-13 Zf Biotox, S.L. Microplaque pour essais biologiques

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CA2063986A1 (fr) 1991-01-29
AU6141690A (en) 1991-03-11

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