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WO2001027626A2 - Moyens permettant d'effectuer des dosages immunologiques et systeme comprenant ces moyens - Google Patents

Moyens permettant d'effectuer des dosages immunologiques et systeme comprenant ces moyens Download PDF

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Publication number
WO2001027626A2
WO2001027626A2 PCT/IL2000/000650 IL0000650W WO0127626A2 WO 2001027626 A2 WO2001027626 A2 WO 2001027626A2 IL 0000650 W IL0000650 W IL 0000650W WO 0127626 A2 WO0127626 A2 WO 0127626A2
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WO
WIPO (PCT)
Prior art keywords
paper
electrode
oxidised
reaction
antigen
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Ceased
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PCT/IL2000/000650
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English (en)
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WO2001027626A3 (fr
Inventor
Yoram Rubin
Nitsa Galili-Nachshon
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BIOPREVENTIVE Ltd
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BIOPREVENTIVE Ltd
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Filing date
Publication date
Priority claimed from IL13241099A external-priority patent/IL132410A0/xx
Priority claimed from IL13241199A external-priority patent/IL132411A0/xx
Application filed by BIOPREVENTIVE Ltd filed Critical BIOPREVENTIVE Ltd
Priority to AU78162/00A priority Critical patent/AU7816200A/en
Publication of WO2001027626A2 publication Critical patent/WO2001027626A2/fr
Publication of WO2001027626A3 publication Critical patent/WO2001027626A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • 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
    • G01N33/88—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving prostaglandins or their receptors

Definitions

  • the present invention relates to means for carrying out amperometric immunoassays with high sensitivity and efficiency particularly, but not exclusively, for the determination of thromboxane,
  • the present invention also relates to a system for carrying out amperometric immunoassays, in particular having a disposable component incorporating such means and a non-disposable component adapted for monitoring the electrical response associated with the immunoassay.
  • Immunoassays are used in the art for the quantitative determination of target substances, having a diagnostic or therapeutic significance, in body fluids.
  • the target substance - antigen - is brought into contact with an antibody that is specific against it or a number of such antibodies and the result of their interaction is measured by any one of the number of methods available in the art.
  • One such method is the amperometric method.
  • TXB 2 thromboxane B 2
  • TXA 2 thromboxane A 2
  • USP 5,047,354 discloses and claims the use of thromboxane to provide an early indication of coronary artery thrombosis by determining the level of TXB 2 or a metabolite thereof or of TXA 2 and comparing said levels with normal levels. Said levels are measured by various methods, e.g., an immunoassay. It is known that immobilisation of the components of immune reaction (i.e., antigens and antibodies) on insoluble support gives significant advantages for their detection in complex mixtures like serum. Immobilised proteins retain their activity over long periods of time.
  • an antibody specific against the antigen that constitutes the target substance and an enzyme, e.g., glucose oxidase (GOX) are immobilised using a glutaraldehyde linker in a polyethylene imine (PEI) layer created on a graphite electrode surface, and brought into contact with a solution consisting of the body fluid to be tested, to which have been added a peroxidase-labeled antibody conjugate and chemicals which form a redox system together with the aforesaid enzyme.
  • PKI polyethylene imine
  • Immobilisation could be achieved by non-covalent adsorption on plastic or on nitrocellulose membranes or by covalent linkages, like immobilisation on CNBr-Sepharose, diazo-derivatives of cellulose or oxidised cellulose.
  • Oxidised celluloses are water insoluble materials produced by reacting cellulose with an oxidant such as gaseous chlorine, hydrogen peroxide, peracetic acid, chlorine dioxide, nitrogen dioxide (dinitrogen tetraoxide), persulfates, permanganate, dichromate-sulfuric acid hypochlorous acid, hyperchlorous acid, hypohalites or periodates.
  • an oxidant such as gaseous chlorine, hydrogen peroxide, peracetic acid, chlorine dioxide, nitrogen dioxide (dinitrogen tetraoxide), persulfates, permanganate, dichromate-sulfuric acid hypochlorous acid, hyperchlorous acid, hypohalites or periodates.
  • oxidised celluloses may contain carboxylic aldehyde and/or ketone functional groups, in addition to the hydroxyl groups, depending on the nature of the oxidant and the reaction conditions used in their preparation.
  • USP 5,780,618 discloses a method for making an oxidised cellulose product from a cellulose material, which involves oxidising a cellulose material into an oxidised cellulose product.
  • USP 4,405,715 discloses enzymes which are immobilised on a solid support material, containing essentially cellulose and lignin, by a process involving oxidation of the support to provide aldehyde groups, animation of the oxidised support by reacting a diamine with the aldehyde groups, reduction of the aminated support to produce stabilized aminated groups, activation of the aminated groups by reacting the groups with a dialdehyde and immobilisation of an enzyme by covalent coupling of the enzyme to the activated groups of the support.
  • a "dipstick" format having a polycarbonate detection membrane fused to a polyvinyl chloride sheet is used to provide an immunoassay method usable under field conditions.
  • C.L. Penny et al., Journal of Immunological Methods, 123: 185-192 ( 1989) describe a method in which the test substance is bound to a detection membrane by immersing such a dipstick into the test sample, which must be a fluid.
  • the dipstick is immersed into a solution containing an antibody, to which is conjugated a means of detection, which is then immobilised by immunological reaction with the bound antigenic substance.
  • the means of detection is executed, which entails immersing the dipstick into a solution containing the appropriate detection reagents.
  • the assay is described as requiring more than one hour to complete, not including pre-assay sample preparation.
  • the apparatus described in the art for effecting immunoassays are laboratory apparatus and are not adapted for use by physicians in hospitals or private clinics, particularly for evaluating patients in the emergency department, rapidly to identify those who require urgent therapy. It is a purpose of the invention to provide a type of solid phase support that is particularly effective for carrying out amperometric immunoassays.
  • the efficiency of the solid phase supports is defined, for the pu ⁇ oses of this invention, by the antigen-binding activity of anti-bodies immobilised therein.
  • the efficiencies of different supports can be compared by comparing the potentials of a given electrode in a given apparatus and in tests of a given solution.
  • the present invention relates to a method of carrying out an amperometric immunoassay of a target antigen, wherein chemicals required for carrying out said assay are provided and immobilised in a solid phase support, characterised in that an oxidised, cellulose paper is provided as said solid phase support.
  • the oxidised cellulose paper is preferably filter paper, particularly filter paper adapted to retain particles of size above 11 ⁇ m.
  • the said oxidised paper preferably comprises a suitable electrode in juxtaposed arrangement therewith.
  • the chemicals required in the assay and immobilised on said paper preferably comprise an enzyme, particularly an oxidase, and an antibody that specifically binds to said target antigen.
  • the method of the invention is preferably used for immunoassaying thromboxane in biological samples.
  • the invention relates to use of oxidised, cellulose paper, preferably filter paper, particularly filter paper adapted to retain particles of size above 1 l ⁇ m, as solid phase support for at least an enzyme and an antibody immobilised thereon in an amperometric immunoassay.
  • the enzyme immobilised on said paper is preferably an oxidase, more particularly glucose oxidase (GOX) and the antigen of interest is thromboxane.
  • GOX glucose oxidase
  • the invention relates to a system for carrying out an amperometric immunoassay of a target antigen comprising:
  • a solid phase support comprising an oxidised cellulose paper comprising immobilised thereon an antibody that specifically binds to said target antigen and at least one enzyme;
  • - receiving means for providing the reaction chamber with a test fluid comprising said target antigen and the chemicals required for the particular immunoassay reaction and a redox reaction occurring in response thereto to be carried out;
  • the said solid phase support, said receiving means, said reaction chamber and said at least one electrode are comprised in a disposable component, and said electronic measuring means are comprised in a non-disposable component, said disposable component being selectively mountable with respect to the non-disposable component such as to provide electrical communication between said at least one electrode and said electronic measuring means.
  • the oxidised cellulose paper is preferably a filter paper, particularly filter paper is adapted to retain particles of size above 1 1 ⁇ m.
  • the system of the invention comprises said at least one electrode, preferably a graphite electrode, juxtaposed with the oxidised paper.
  • the electronic measurement means in the system of the invention comprises suitable amperometric circuit means for measuring an electric potential of the said at least one electrode.
  • the electronic measurement means may further comprise means for digitally processing a change in said electric potential generated in response to said reaction to provide a signal representative of the immunoassay.
  • the system of the invention may further comprise suitable memory means for storing comparison values used for enabling calculation of a desired parameter representing the result of the assay from said signal, preferably target concentration data, and processing means for carrying out such calculation.
  • system of the invention may comprise a suitable electronic display adapted for displaying said parameter, display preferably being comprised in the said non-disposable component.
  • the electrode in the system of the invention may comprise an outwardly extending tip with respect to said disposable component.
  • the said electronic measurement means may comprise a terminal operatively connected thereto and adapted for engaging with said tip such as to establish electrical communication therewith when said disposable component is mounted to said non-disposable component.
  • the disposable component comprises a suitable vessel having a first chamber adapted for receiving a sample containing said target antigen, said chemicals required for the particular immunoassay reaction and said redox reaction, and a substrate of the enzyme immobilised on said oxidised cellulose paper and a second chamber in fluid communication with the first chamber, said second chamber being said reaction chamber and comprising said paper and said at least one electrode.
  • the said disposable part of the system of the invention preferably comprises a housing having at least three reservoirs each one for separately holding a desired liquid, each reservoir comprising an outlet selectively in communication with said reaction chamber via selection means to enable at least one of said at least three liquids to be selectively introduced to the reaction chamber, said housing further comprising a suitable slot for accommodating therein said paper and said electrode.
  • the said selection means may comprise a suitable cylinder mounted for rotation within said housing, said cylinder having an open end within the housing facing said paper, and a closed end having external engagement means enabling rotation of the cylinder by external manipulation of said engagement means, wherein said cylinder comprises apertures in staggered circumferential arrangement such as enable one aperture at a time to be selectively brought into registry with a corresponding outlet of a said reservoir to provide communication between the reservoir and the reaction chamber, in response to a corresponding angular displacement of the cylinder with respect to the housing.
  • the said liquid in one of said reservoirs is the test liquid comprising the target antigen, a liquid comprising suitable chemicals required for the particular immunoassay reaction and a redox reaction or a liquid comprising a substrate of the enzyme immobilised on said paper.
  • the reagent required for the immuno-reaction preferably comprise comprises an oxidase-labeled antibody-antigen conjugate, wherein the antigen is the same as the analyte of interest, preferably horseradish peroxidase-labeled antibody-antigen conjugate.
  • the enzyme immobilised on said paper is GOX
  • the components required to induce a redox reaction with the aforesaid oxidase may comprise glucose.
  • system of the invention may further comprise a suitable porous member inte ⁇ osed between said paper and said reaction chamber, said porous member capable of providing fluid communication therebetween.
  • the porous member preferably comprises at least 0.5 cc of voids per 1 cc of volume.
  • system of the invention may comprise suitable power means, for example at least one electrical battery.
  • the system of the invention is preferably used for determination of TXB 2 or its metabolites or those of TXA 2 , in which case the antibody immobilised on said paper is capable of specifically binding to TXB 2 or its metabolites or those of TXA 2 .
  • glucose oxidase will be referred to hereinafter as a preferred example.
  • various electrodes can be used, but a preferred embodiment is a graphite electrode.
  • One aspect of the invention is a method of carrying out amperometric assays, particularly immunoassays, which comprises providing chemicals required in said assay, particularly an enzyme, e.g. but not exclusively an oxidase, e.g. glucose oxidase (GOX), and an antibody specific against the target antigen, and immobilising them in an oxidised cellulose paper support.
  • an enzyme e.g. but not exclusively an oxidase, e.g. glucose oxidase (GOX)
  • GOX glucose oxidase
  • the remaining features of the assay will be assumed, for the pu ⁇ ose of illustration, to be those described by Rishpon et al., but it will be understood that the invention is not limited to such features, but extends to immobilising an enzyme and an antibody in an oxidised paper support for amperometric assays.
  • the invention further extends to any amperometric assay that comprises monitoring the potential of an electrode resulting from a reaction that involves reagents immobilised in a solid phase support.
  • a preferred type of oxidised paper is obtained by oxidising filter paper, particularly filter paper that retains particles above 1 l ⁇ m. Oxidising processes are known in the art and can be applied to carry out the invention. A preferred method is that described in the article by Gurvich A.E and Lechtzind, E.V., "High capacity immunoabsorbents based on preparations of reprecipitated cellulose", Lab. Chem. Biosynth. Antibodies, Gamaleya Inst. Epidemiol. Microbiol., Moscow, 123098, USSR Mol. Immunol. (1982), 19(4), 637-40, the contents of which are inco ⁇ orated herein by reference.
  • a further aspect of the invention is the use of oxidised paper for immobilising substances participating in an amperometric assay, particularly immunoassay.
  • Said substances may be particularly an enzyme and an antibody.
  • FIG. 1 illustrates in upper front perspective view the disposable part of a system according to the first embodiment of the invention.
  • Figure 2 illustrates in upper rear perspective view the embodiment of Figure 1.
  • FIG. 3 illustrates in upper front perspective view the non-disposable part of the system according to the first embodiment of the present invention.
  • Figure 4 illustrates in upper front perspective view the disposable part of Figures 1 and 2 mounted to the non-disposable part of Figure 3.
  • Figure 5 illustrates in upper rear perspective view the embodiment of Figure 4.
  • FIG. 6 illustrates in upper front perspective view a system according to a second embodiment of the present invention.
  • FIG. 7 illustrates in upper front perspective view the disposable part of the embodiment of Figure 6.
  • FIG. 8 illustrates in perspective cross-sectional view the embodiment of Figure 6.
  • FIG. 10 illustrates in perspective cross-sectional view the disposable part according to a third embodiment of the present invention.
  • Figure 1 1 graphically represents the electric current generated in a kit according to the present invention as a function of time, obtained in immunoassays of thromboxane B for various concentrations thereof in the tested body fluid.
  • the present invention relates to means for carrying out amperometric immunoassays with particular sensitivity and efficiency, said means being typically in the form of a solid phase support for immobilising a specific antibody and an enzyme.
  • the said solid state support according to the present invention comprises an oxidised, cellulose paper.
  • the present invention also relates to a system for performing amperometric immunoassays, comprising a disposable part selectively mountable to an apparatus.
  • the disposable part comprises said means for carrying out the immunoassay, and a non-disposable part for analysing and displaying the results of the assay, as is described hereinbelow in greater detail.
  • the oxidised paper has covalently immobilised therein GOX or a similar oxidase, capable of generating hydrogen peroxide from a suitable substrate, and an antibody that is capable of specifically binding to the antigen of interest to be assayed.
  • GOX peroxidase-labeled antibody-antigen conjugate
  • the antigen is the same as the analyte of interest, as well as the other components required to create a redox system with the aforesaid oxidase, e.g., aminosalicylic acid, and glucose and iodide ions.
  • the GOX catalyzes the oxidation of glucose to
  • H 2 0 2 H 2 0 2 .
  • Iodine is produced in the peroxidase-catalyzed H 0 2 /iodide redox system. If the antigen of interest is absent from the sample, the antibody immobilised on the oxidised paper binds only the peroxidase-labeled antibody-antigen conjugate, and the degree of said reaction is measured amperometrically by the electrochemical reduction of iodine back to iodide. If there was antigen present in the sample, this unconjugated antigen competes with the peroxidase-labeled antibody-antigen conjugate for the available sites of the antibody immobilised on the oxidised paper; or, alternatively, displaces the antigen from the conjugate. In these cases, the degree of said reaction is correspondingly lower and the amount of antigen in the tested liquid can be calculated from the reduction of said degree. Said method, however, is mentioned by way of illustration only and does not constitute a limitation to the use of the present
  • peroxidase-labeled antibody may be used in place of the peroxidase-labeled antibody-antigen described above.
  • the antigen present in the sample is bound by one or more epitopes to the antibody immobilised on the oxidised paper, and by different epitopes to a peroxidase-labeled antibody.
  • the amperometric signal increases with increasing concentrations of antigen in the liquid sample.
  • a preferred example of the use of the system of the invention is the determination of TXB 2 or its metabolites or metabolites of TXA 2 , that are contained in a patient's urine.
  • the system is usable for any other immunoassay, with the provision of the appropriate antibody and optionally enzyme are immobilised on the oxidised paper.
  • the oxidised paper is typically prepared with the antibody and enzyme either before the assay.
  • Example 1 Oxidised Paper
  • Filter paper retaining particles above 1 1 ⁇ m, was used as starting material. Its oxidation was performed as follows: Paper strips 1.5 cm width were incubated overnight in 0.01 KI0 4 (2.14 g/1) in dark at room temperature. The next day, the strips were washed 5 times by distilled water and kept in water in a refrigerator. Binding activity remained unchanged during storage in water at least one to two years. Due to the oxidation, a number of glucose rings is cleaved and two aldehyde groups per each ring appear. Protein molecules are reacted by their amino groups with aldehyde groups in alkali solution and covalently as well firmly bind to cellulose.
  • Strips of oxidised paper, obtained as in Example 1 were soaked for 15 minutes in O. l M carbonate-bicarbonate buffer pH 9 (2.756 g/1 Na 2 C0 3 + 6.216 g/1 NaHC0 3 ). After drying on filter paper 10 ⁇ of protein solution made in the same buffer was applied on paper by automatic pipette. The size of spots was about 1 cm in diameter. Strips were placed on thin glass tubes in a Petri dish and incubated for two hours in wet conditions at room temperature. After washing with phosphate-buffered saline (PBS) (5 min, five times at rotation), paper strips with immobilised proteins can be stored in the refrigerator in PBS for a long time.
  • PBS phosphate-buffered saline
  • monoclonal anti-IgG antibodies retained their capacity to bind antigen after immobilisation on oxidised paper.
  • Five ⁇ g of immobilised antibodies were able to adsorb specifically about 3 ⁇ g of human IgG as antigen.
  • the same amount of immobilised antibodies could adsorb about 7.5 ng of a small molecular weight antigen like thromboxane B 2 with molecular weight 370 Da.
  • Such capacity is enough for using immunoadsrbents based on oxidised paper for preparation of diagnostic dipsticks.
  • oxidised paper was prepared by incubating filter paper in 100 ml of 0.01M NaI0 overnight (ON) at room temperature
  • Peroxidase reaction Drain excess buffer of the paper on kimwipe. Add peroxidase substrate - TMB (precipitating) to the paper; use 100 ⁇ l for 1 cm". Stop the reaction when a blue spot appears (up to 15 min.) by washing twice with DW (-1/2 hr). Document results visually and by colorimeter measurements.
  • oxidised paper as solid phase support of chemicals used in immunoassays, particularly enzymes and antibodies, provides an important improvement on the use of polymer coatings or layers, e.g. PEI, as described by Ivnitzki et al. and Rishpon et al., in that they generate a stronger and sha ⁇ er reaction of the immunoassay electrode. This is demonstrated by Example 5.
  • PEI polymer coatings or layers
  • a solution containing 0.22 ng/1 of thromboxane B 2 and the reagents of the aforesaid "Thromboxane B 2 enzyme immunoassay (EIA) system” was contacted with a graphite electrode coupled with filter paper obtained as in Example 1.
  • the current generated in a conductor connecting the electrode to ground was monitored after 650 seconds. Said current increased at a rate of 0.17 nA/sec. If the same solution is contacted with an electrode as described in Rishpon et al, no current increase is detected.
  • the present invention also relates to a system for performing immunoassays, comprising a disposable part selectively mountable to an apparatus.
  • the disposable part is adapted for actually carrying out the immunoassay, and comprises a reaction chamber and suitable means for introducing thereinto the fluid to be tested, that comprises the antigen of interest, that is the target substance, as well as for introducing any chemicals required for conducting the immuno-reaction and a redox reaction.
  • the disposable part also comprises an oxidised cellulose paper membrane according to the present invention, adapted to be in contact with the contents of the reaction chamber, and juxtaposed with a suitable electrode.
  • the paper may be previously prepared with an appropriate enzyme and antigen for a specific test immobilised thereon.
  • the electrode is selectively operatively connectable to the apparatus when the reaction chamber is mounted thereon, and the apparatus is adapted for monitoring the response of the electrode to a redox reaction that occurs in the reaction chamber.
  • the disposable part is typically in the form of a cartridge that is selectively mountable and dismountable with respect to apparatus, which in turn is adapted for enabling any one of a number of disposable cartridges to be mounted thereto and dismounted therefrom, one at a time.
  • the disposable part is made from a material and in such a manner as to constitute a relatively small or insignificant economic outlay, particularly with respect to the apparatus itself, and is thus discarded after one test.
  • the apparatus monitors the response of the electrode via suitable electronic measuring means, which is in operative electrical contact with the electrode when the system is in operation and the disposable part is mounted on the apparatus.
  • the electronic measuring means typically includes suitable circuit means for generating from the electrode potential an electric current and measuring its intensity, and digital elaborating means, comprising a memory and a CPU programmed for deriving from said current intensity a signal representing the parameter (e.g. the antigen concentration) that represents the results of the assay. This generally requires storing in said memory a set of comparative or zero data.
  • the device should display the concentration of the target substance from the electrode potential and display its value
  • disposable is herein taken to mean "designed to be thrown away after use with only negligible loss".
  • the useful life of the disposable part ends with either contamination of the contents therein, or with reaction of the contents within the reaction chamber.
  • dispenser is clearly understood in the context of the present specification to refer to the disposable part being designed to be thrown away after use with negligible economic loss, after completion of the immunoassay.
  • the non-disposable apparatus of the present invention is designed for many use cycles, each cycle with a new disposable part mounted thereto, rather than being designed to be disposed of the first time that it is used, and may thus constitute a significant capital item in relation to the disposable part.
  • reaction chamber is meant herein the space or volume comprised in the disposable part that is adapted for enabling the fluid to be tested to come into contact with the oxidised paper and with the electrode.
  • FIG. 1 to 5 illustrate a first embodiment of the present invention.
  • the system designated by the numeral ( 100), comprises a disposable part (10) and a non-disposable monitoring apparatus (20).
  • the disposable part (10) is typically in the form of a cartridge, and is selectively mountable and dismountable with respect to said apparatus (20).
  • the disposable part (10) comprises a vessel (1 1) adapted for receiving the body fluid to be tested, as well as any chemicals required for conducting the immunoassay reaction and the redox reaction.
  • the vessel (1 1) may be in the form of container having peripheral wall ( 16) including a back wall (18) joined to a base (17), preferably integrally, to define a first volume, and having an open upper end (which may be optionally closeable with a suitable lid) via which the fluid to be tested may be introduced into the first volume.
  • the vessel (1 1) further comprises a reaction chamber (12), typically juxtaposed with the back wall (18), the reaction chamber (12) being in fluid communication with the first volume in the vessel via one or more suitable openings (15).
  • the reaction chamber (12) is adapted for accommodating oxidised paper (13) and a vertical portion of electrode (14) that is in juxtaposition with the paper (13).
  • the electrode is typically in the form of an inverted "L", having in addition to the vertical portion a horizontal portion that extends backwardly from the vessel (11), accommodated on a tab (19) that also extends in a backward direction from back wall (18).
  • the vessel (1 1) may be made from any suitable thin plastic or paper, for example, advantageously as a moulded product.
  • the non-disposable apparatus (20) comprises a housing portion (22) with a tongue-like pedestal (21) extending outwardly from a lower end thereof.
  • the pedestal (21) has an upper surface adapted for releasable engagement with the base ( 17) to enable the disposable part ( 10) to be easily mounted and then dismounted from the apparatus (20) after use.
  • the upper part of pedestal (21) is advantageously contoured in complementary manner with respect to the lower surface of base (17).
  • the housing portion (22) also comprises a forward facing wall (27), typically complementary at least in part to back wall (18) of the vessel (1 1), and comprises a slot (26) adapted for receiving the tab (19) and the part of the electrode (14) accommodated thereon when the disposable part (10) is mounted onto the apparatus (20).
  • Slot (26) comprises at least one suitable electrical terminal of suitable electronic measuring means (not shown) also accommodated in the housing (22).
  • the electronic measuring means are adapted for amperometrically measuring the potential of the electrode (14), as hereinbefore described.
  • the housing (22) further comprises an on/off switch or button (23) for activating/deactivating respectively the electronic measuring means.
  • the housing (22) also comprises a display (24), typically an LCD display or the like, for showing the results of the immunoassay including information relating to target substance concentration or any other associated or desired information, typically in alphanumeric, graphical or any other suitable form.
  • the apparatus (20) is typically portable, and comprises a suitable battery (25).
  • a suitable DC or AC lead (not shown) for connection to a suitable power source may be provided.
  • the cartridge-like disposable part (10) is mounted to the apparatus (20) such that the horizontal part of electrode (14) is inserted into slot (27), establishing operational electrical contact with the electronic measuring means in housing (22).
  • Body fluid to be tested for the target substance typically an antigen
  • the chemicals required for the immunoassay reaction and the redox reaction are similarly introduced into the vessel (11) and thus into the reaction chamber (12). Electrical changes associated with the immunoassay-induced redox reaction are detected via the electrode (14) and transmitted to the electronic measuring means in the apparatus (20) for analysis and display.
  • FIG. 6 A second embodiment of the system (200) according to the present invention is illustrated in Figures 6 to 9.
  • the disposable part (30) of the system (200) is in the form of a housing (33) having a horizontal bore for accommodating therein a cylindrical member (43).
  • the cylindrical member (43) is open at one end thereof, and closed at the other end thereof, comprising thereat a knob (34) which protrudes outside a front end (50) of the housing (33). External manipulation of the knob (34) enables the cylinder (43) to be turned with respect to the bore and thus the housing (33).
  • the housing (33) further comprises three vertically disposed reservoirs or internal passages (32) for separately introducing and holding the liquid to be tested, the enzyme-labeled antibody-antigen conjugate, preferably peroxidase-labeled antibody-antigen conjugate and the chemical reagent/s required for the redox reaction, for example aminosalicylic acid, and the substrate of the enzyme immobilised on the oxidised paper, into the cylinder (43).
  • the cylinder (43) comprises three angularly staggered openings (36) spaced one from the other longitudinally such that on rotating the cylinder (43) only one of the openings (36) at a time is brought into registry with corresponding passages (32).
  • fluid communication may be selectively established between the inner portion of the cylinder, which constitutes the reaction chamber (56) and each one of the passages (32) separately, enabling the different fluids to be delivered to the reaction chamber (56) at different times.
  • more than 3 passages (32), or indeed less than 3 passages (32) may be provided, as required, mutatis mutandis.
  • the open longitudinal end of the cylinder (43) is in open communication with a vertical well or slot (35) which extends to the upper end of the housing (33).
  • the slot is adapted to accommodate therein an oxidised paper (38) according to the present invention, preferably in the form of a strip, the oxidised paper (38) being juxtaposed with a suitable electrode (39).
  • the oxidised paper (38) is in communication with the reaction chamber (56). At least the electrode (39) extends upwardly beyond the upper part of the housing (33). Part of the oxidised paper (38) is in communication with the reaction chamber (56) via the open end of cylinder (43). The fluids provided to the reaction chamber (56) via passages (32) come into contact with the oxidised paper (38) and through it with the juxtaposed electrode (39). The part of the electrode (39) that extends upwardly from the housing (33) is receivable into a downwardly facing slot (48) comprised into the non-disposable apparatus (40) when the disposable part (30) is mounted thereto, enabling electrical contact to be re ⁇
  • the apparatus (40) preferably comprises one or more batteries (41) for portability, though may optionally or alternatively comprise a suitable lead for connection to an external AC or DC source.
  • the apparatus (40) also preferably comprises a display (42), visible through an opening (45).
  • the disposable part (30) is mountable to the apparatus (40) by means of sliding engagement of rail member (53) formed at the back end (52) of the disposable part (30), with respect to a complementary slot (not shown) in the apparatus (40).
  • a third embodiment of the present invention comprises the same structural elements as the second embodiment, with the exception of the said slot (35), as hereinbefore described, mutatis mutandis.
  • a deeper slot (35') is provided, enabling a porous member (44), typically in the form of a rectangular strip, to be inte ⁇ osed between the oxidised paper (38) and the reaction chamber (56).
  • a porous member (44) typically in the form of a rectangular strip
  • the liquids contained in the reaction chamber (56) progress by capillarity along said porous member (44) and, through the oxidised paper (38) come into contact with the electrode (39).
  • the porous member (44) provides advantages in the operation of the system.
  • the porous member (44) is advantageously comprised of a spongy body of any suitable material, e.g. a material such as polystyrene, having a volume of voids of at least 0.5 cc per lcc.
  • the oxidised paper is preferably, though not necessarily, filter paper such as will retain any particles having a dimension above 1 1 ⁇ m. Oxidation of the paper can be carried out by processes known in the art, e.g. by incubating with 0.0 IM solution of KI0 4 for a number of hours in the dark at room temperature.
  • Figure 1 1 graphically represents the current developed in a kit or system according to the invention used for the immunoassay of thromboxane B 2 in liquids of various concentrations from 0.00 to 0.66 n/1, as well as for a blank assay, i.e., an assay in which the liquid did not contain an enzyme nor an antibody.
  • Figure 12 the gradients of the parts of the curves of Figure 1 1 referring to times higher than 650 seconds, are shown in a comparative bar-chart.
  • One such parameter can be the conductivity of the solution, which may provide additional useful information.
  • an additional electrode To measure such a parameter, an additional electrode must be provided and a potential applied to it by the battery of the kit. This will cause a current to flow from the additional electrode to the electrode of the embodiments of Figs. 1 to 10 and said current may be measured amperometrically by the same electronic circuit of the previous embodiments or another one adapted for this pu ⁇ ose. Skilled persons can provide the kit with additional components, should other physical parameters of the tested fluid be measured.
  • the embodiments of the system according to the present invention have been described as comprising a disposable part selectively mountable to a non-disposable apparatus, and such an arrangement enables a plurality of tests to be conducted using the same apparatus, though using a new, sterilised, disposable part for each individual test, to be disposed of thereafter.
  • the disposable part may in fact be partly reusable, or fully reusable, being re-sterilisable.
  • the oxidised paper would need to be replaced between tests.
  • these could be integrally formed within the apparatus itself in a suitable manner.

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

Abstract

L'invention concerne un système et un procédé associé permettant d'effectuer un dosage immunologique ampérométrique d'un antigène cible. Ce système comprend un support en phase solide comprenant un papier de cellulose oxydée sur lequel est immobilisé un anticorps qui se lie spécifiquement à l'antigène cible, et au moins un enzyme. Une électrode est juxtaposée au papier, lequel communique avec une chambre de réaction présentant des moyens récepteurs adéquats permettant l'introduction d'un liquide de test contenant l'antigène cible ainsi que des produits chimiques nécessaires à la réaction de dosage immunologique spécifique, et à une réaction d'oxydoréduction se produisant en réponse à celle-ci. Des moyens de mesure électroniques adéquats sont connectés de manière opérationnelle avec l'électrode afin de surveiller la réponse de cette (ces) électrode(s) à la réaction d'oxydoréduction se produisant dans la chambre de réaction. De manière spécifique, la chambre de réaction, les moyens récepteurs, le papier et l'électrode se présentent sous forme d'une cartouche jetable qui peut être monté sur des moyens de mesure électroniques réutilisables pendant l'opération de dosage.
PCT/IL2000/000650 1999-10-01 2000-10-12 Moyens permettant d'effectuer des dosages immunologiques et systeme comprenant ces moyens Ceased WO2001027626A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU78162/00A AU7816200A (en) 1999-10-01 2000-10-12 Means for carrying out immunoassays and system incorporating same

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
IL13241099A IL132410A0 (en) 1999-10-01 1999-10-01 Means for carrying out immunoassays
IL13241199A IL132411A0 (en) 1999-10-14 1999-10-14 Disposable kit for immunoassays
IL132411 1999-10-14
IL132410 1999-10-14

Publications (2)

Publication Number Publication Date
WO2001027626A2 true WO2001027626A2 (fr) 2001-04-19
WO2001027626A3 WO2001027626A3 (fr) 2001-10-18

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Country Link
AU (1) AU7816200A (fr)
WO (1) WO2001027626A2 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004065951A1 (fr) * 2003-01-20 2004-08-05 Universal Biosensors Pty Limited Procede de detection electrochimique
WO2006087697A3 (fr) * 2005-02-15 2007-05-24 Analyte Works Ltd Dispositif de test permettant d'examiner des echantillons de fluide, notamment d'urine
US8507260B2 (en) 2005-01-31 2013-08-13 Realbio Technologies, Ltd. Multistep reaction lateral flow capillary
US9726581B2 (en) 2011-12-22 2017-08-08 Realbio Technologies Ltd. Sequential lateral flow capillary device for analyte determination
US9952211B2 (en) 2008-06-29 2018-04-24 Realbio Technologies Ltd. Liquid-transfer device particularly useful as a capturing device in a biological assay process
US20230333118A1 (en) * 2019-09-20 2023-10-19 Momm Diagnostics Gmbh Immunoassay analyzer, immunoassay kit and method for detecting analyte in liquid sample
EP4343316A4 (fr) * 2022-06-20 2024-11-06 Shanghai United Imaging Microelectronics Technology Co., Ltd. Utilisation d'un composé polymère naturel dans un biodétecteur

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2472015A1 (fr) * 1979-12-21 1981-06-26 Beghin Say Sa Enzymes immobilisees sur un support solide
US5047354A (en) * 1987-10-30 1991-09-10 Foegh Marie L Thromboxane B2 assay for coronary artery thrombosis
US5015387A (en) * 1988-10-18 1991-05-14 Fuji Photo Film Co., Ltd. Method for activating cellulosic membrane, activated cellulosic membrane, method of fixing physiologically active substance on the activated cellulosic membrane and physiologically active substance-fixed membrane
JPH06507709A (ja) * 1991-06-04 1994-09-01 ファイスンズ　ピーエルシー 分析装置
WO1997045741A1 (fr) * 1996-05-29 1997-12-04 Wilkins Ebtisam S Immunoelectrode dans l'ecoulement du fluide pour immunodosage amperometrique rapide
GB9623147D0 (en) * 1996-11-07 1997-01-08 Univ Manchester Membrane for a sensor
IES80556B2 (en) * 1997-01-10 1998-09-23 Robert Arpad Foster Electrochemical diagnostic detection device

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7403017B2 (en) 2003-01-20 2008-07-22 Universal Biosensors Pty Limited Methods of measuring barrier formation
CN100458430C (zh) * 2003-01-20 2009-02-04 环球生物传感器有限公司 电化学检测方法及设备
WO2004065951A1 (fr) * 2003-01-20 2004-08-05 Universal Biosensors Pty Limited Procede de detection electrochimique
US8507260B2 (en) 2005-01-31 2013-08-13 Realbio Technologies, Ltd. Multistep reaction lateral flow capillary
US9101927B2 (en) 2005-01-31 2015-08-11 Realbio Technologies Ltd. Multistep reaction lateral flow capillary device
US10335783B2 (en) 2005-01-31 2019-07-02 Realbio Technologies, Ltd. Multistep reaction lateral flow capillary device
WO2006087697A3 (fr) * 2005-02-15 2007-05-24 Analyte Works Ltd Dispositif de test permettant d'examiner des echantillons de fluide, notamment d'urine
US11280785B2 (en) 2008-06-29 2022-03-22 Realbio Technologies Ltd. Liquid-transfer device particularly useful as a capturing device in a biological assay process
US9952211B2 (en) 2008-06-29 2018-04-24 Realbio Technologies Ltd. Liquid-transfer device particularly useful as a capturing device in a biological assay process
US9726581B2 (en) 2011-12-22 2017-08-08 Realbio Technologies Ltd. Sequential lateral flow capillary device for analyte determination
US10598572B2 (en) 2011-12-22 2020-03-24 Realbio Technologies, Ltd. Sequential lateral capillary flow device for analyte determination
US20230333118A1 (en) * 2019-09-20 2023-10-19 Momm Diagnostics Gmbh Immunoassay analyzer, immunoassay kit and method for detecting analyte in liquid sample
EP4343316A4 (fr) * 2022-06-20 2024-11-06 Shanghai United Imaging Microelectronics Technology Co., Ltd. Utilisation d'un composé polymère naturel dans un biodétecteur

Also Published As

Publication number Publication date
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WO2001027626A3 (fr) 2001-10-18

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