WO2001027626A2

WO2001027626A2 - Means and system for carrying out immunoassays

Info

Publication number: WO2001027626A2
Application number: PCT/IL2000/000650
Authority: WO
Inventors: Yoram Rubin; Nitsa Galili-Nachshon
Original assignee: Biopreventive Ltd.
Priority date: 1999-10-01
Filing date: 2000-10-12
Publication date: 2001-04-19
Also published as: WO2001027626A3; AU7816200A

Abstract

A system and corresponding method for carrying out an amperometric immunoassay of a target antigen in which a solid phase support is provided, having an oxidised cellulose paper on which is immobilised an antibody that specifically binds to target antigen and at least one enzyme. An electrode is juxtaposed with the paper, which is in communication with a reaction chamber having suitable receiving means for providing thereto a test fluid having said target antigen and the chemicals required for the particular immunoassay reaction and a redox reaction occurring in response thereto to be carried out. Suitable electronic measuring means are operatively connected to the electrode for monitoring a response of said at least one electrode to said redox reaction that occurs in the reaction chamber. In particular, the reaction chamber, receiving means, paper and electrode are in the form of a disposable cartridge which is mountable to a separate non-disposable electronic measuring means when the assay is to be performed.

Description

MEANS FOR CARRYING OUT IMMUNOASSAYS AND SYSTEM

INCORPORATING SAME

Field of the Invention

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.

Background of the Invention

Immunoassays are used in the art for the quantitative determination of target substances, having a diagnostic or therapeutic significance, in body fluids. For this purpose, 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.

One particular antigen to which the art refers is thromboxane B₂ (TXB₂) or a metabolite thereof or of thromboxane A₂ (TXA₂). 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 TXB₂ or a metabolite thereof or of TXA₂ 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.

D. Ivnitzki and J. Rishpon in "A one-step, separation-free amperometric enzyme immunosensor", Biosensors & Bioelectronics, Vol. 1 1, No. 4, pp. 409-417, Elsevier Science Limited, 1996, describe a one-step, separation-free, amperometric enzyme immunosensor which comprises an antibody electrode and an amplification system. The immunological reactions are monitored electrochemically in situ, and the binding curves are directly visualized on a computer screen. This reference will be referred to hereinafter as Ivnitzki et al.

J. Rishpon and D. Ivnitzki in "Amperometric Enzyme-channeling Immunosensor", Enzyme Engineering XIII, Vol. 799 of the Annals of the New York Academy of Science likewise describe enzyme immunoassays (EIA) and apparatus for carrying them out. This reference will be referred to hereinafter as Rishpon et al.

In these immunoassays, 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. While these are specific examples of immunoassays, different ones can be carried out, in which an enzyme and an antibody are preferably immobilised on a solid phase support.

It has been found that the efficiency of the immunoassays is sharply dependent on the type of solid phase support used for immobilising the enzyme and the antibody.

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 (or oxycelluloses) 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. These 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. The preparation of oxidised celluloses suitable for use as carrier vehicles in the development of cosmetic and pharmaceutical preparations, using alkali metal or alkaline earth metal hypohalites, is discussed in USP 4,480,089. Also, 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.

Immunoassays have been described in US Patents 4,447,564, 4,654,532,

4,716,121 and 4,582,809, and WO 93/20240, which are all incorporated herein by reference. In one example, 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. In a second step, 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. In a final step, 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.

Additionally, it is another purpose of the invention to provide a type of solid phase support that is adapted for use in disposable or partly disposable immunoassay kits.

It is further purpose of the invention to provide a type of solid phase support that is easily prepared and inexpensive.

It is yet a further purpose of this invention to provide a system and apparatus, preferably in kit-form, that permits rapid determination by immunoassays of target substances having diagnostic or therapeutic significance.

It is another purpose of this invention to provide such an apparatus which constitutes an amperometric enzyme immunosensor.

It is a further purpose of this invention to provide such an apparatus that is particularly useful for the determination of thromboxane or its metabolites.

It is a still further purpose of this invention to provide such an apparatus which is partly disposable, comprising a first part that can be used for only one test and then discarded, while another part, of greater complication and cost, can be retained and used repeatedly.

It is a still further purpose to provide such an apparatus that is simple, of safe operation and inexpensive. It is a still further purpose to provide such an apparatus that permits determination of other physical parameters of body fluids, in particular conductivity.

Other puφoses and advantages of the invention will appear as the description proceeds.

The efficiency of the solid phase supports is defined, for the puφoses of this invention, by the antigen-binding activity of anti-bodies immobilised therein. For the particular puφose of amperometric immunoassays, 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.

Summary of the Invention

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 μm.

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.

In a further embodiment, the invention relates to use of oxidised, cellulose paper, preferably filter paper, particularly filter paper adapted to retain particles of size above 1 l μm, as solid phase support for at least an enzyme and an antibody immobilised thereon in an amperometric immunoassay. Also in this embodiment, the enzyme immobilised on said paper is preferably an oxidase, more particularly glucose oxidase (GOX) and the antigen of interest is thromboxane.

In yet a further embodiment, 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;

- reaction chamber in communication with said paper;

- 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;

- at least one electrode juxtaposed with said paper; and - suitable electronic measuring means operatively connected to said at least one electrode for monitoring a response of said at least one electrode to said redox reaction that occurs in the reaction chamber.

In a specific embodiment of the system of the invention, 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.

Also in the system of the invention, the oxidised cellulose paper is preferably a filter paper, particularly filter paper is adapted to retain particles of size above 1 1 μm.

In preferred embodiments, 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. Further, 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.

Further, the 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.

Still further, 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.

In a preferred embodiment of the system of the invention, 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. When the enzyme immobilised on said paper is GOX, the components required to induce a redox reaction with the aforesaid oxidase, may comprise glucose.

Still more particularly, the system of the invention may further comprise a suitable porous member inteφosed 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.

Further, the 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 TXB₂ or its metabolites or those of TXA₂, in which case the antibody immobilised on said paper is capable of specifically binding to TXB₂ or its metabolites or those of TXA₂.

While various enzymes to be immobilised on the said paper can be used, glucose oxidase will be referred to hereinafter as a preferred example. Likewise, 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. The remaining features of the assay will be assumed, for the puφose 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 μm. 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 incoφorated herein by reference. While in said article reprecipitated cellulose is described as the starting material, and porous spherical beads are obtained, it has been found that the same treatment can be applied to paper, particularly filter paper, and the oxidised product thus obtained is a preferred material according to this invention.

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. Brief Description of the Drawings

In the drawings:

- Figure 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.

- Figure 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.

- Figure 6 illustrates in upper front perspective view a system according to a second embodiment of the present invention.

- Figure 7 illustrates in upper front perspective view the disposable part of the embodiment of Figure 6.

- Figure 8 illustrates in perspective cross-sectional view the embodiment of Figure 6.

- Figure 9 illustrates in perspective cross-sectional view the embodiment of Figure 7.

- Figure 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.

- Figure 12 illustrates in bar-chart form the gradients of the curves of Figure 1 1. Detailed Description of Preferred Embodiments

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. In particular, 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.

In one type of immunoassay method, for which the system of the invention is typically used, 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. To the sample tested there are added a peroxidase-labeled antibody-antigen conjugate, wherein 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

H₂0₂. Iodine is produced in the peroxidase-catalyzed H 0₂/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 invention.

In another mode of operation, peroxidase-labeled antibody may be used in place of the peroxidase-labeled antibody-antigen described above. In this "sandwich" type of assay, 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. In this mode of operation, the amperometric signal increases with increasing concentrations of antigen in the liquid sample.

As stated hereinbefore, a preferred example of the use of the system of the invention is the determination of TXB₂ or its metabolites or metabolites of TXA₂, that are contained in a patient's urine. However, 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.

The following examples illustrate, but do not limit the method and system according to the present invention. Example 1 - Oxidised Paper

Filter paper, retaining particles above 1 1 μm, was used as starting material. Its oxidation was performed as follows: Paper strips 1.5 cm width were incubated overnight in 0.01 KI0₄ (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.

The ability of the oxidised paper thus obtained to act as an immobilising support is evidenced by the following test.

Example 2 - Immobilisation of Proteins

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 Na₂C0₃ + 6.216 g/1 NaHC0₃). After drying on filter paper 10 Φμ 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. To determine the immobilisation capacity of said oxidised paper, 10 ml of human IgG solutions of different concentrations were applied on oxidised paper strips obtained as in Example 1. After incubation and washing from non-bound protein, strips were stained in a solution of Coomassie dye (0.02% in 50% ethanol) for 15 min and after washing with de-staining solution (5% methanol in 7% acetic acid) the dye bound to protein was eluted by 2 ml 50% acetic acid. The absorbencies of eluates were measured at 590 nm. The amount of immobilised IgG was determined using a calibration curve. The curve was obtained by applying known amounts of IgG on non-oxidised paper. After incubation in 5% trichloroacetic acid for 15 min, paper strips were stained by Coomassie and absorbency of eluted dye was measured at 590 nm. The capacity of oxidised paper to immobilised IgG (purified on DEAE cellulose ion exchange resin) is shown in Table I.

From these data, it is demonstrated that the immobilisation capacity of oxidised paper depended on the amount of applied protein. On a spot of 1 cm diameter, about 15 μg of proteins can be immobilised.

Table I: Capacity of Oxidised Paper to Immobilise Proteins

Example 3 - Antigen-Binding Activity of Antibodies Immobilised on Oxidised Paper

To study the activity of immobilised antibodies, the system of human IgG and monoclonal anti-IgG antibodies was used. Commercial ascite fluid, which contain monoclonal anti-IgG antibodies was diluted 1 : 1 with 0.1M carbonate-bicarbonate buffer, pH 9.2 and applied on oxidised paper. As antigen, a preparation of biotinylated human IgG was used. For calculation of the amount of IgG reacted with immobilised antibodies, the calibration curve was obtained using different amounts of biotinylated IgG applied on non-oxidised paper. Paper strips were treated with Streptavidin (1 :25,000) (Amersham, England) for 30 min. After washing 15 min with PBS, strips were covered wi+th enhanced chemiluminescence detection reagent (ECL) for 1 min. X-ray film was placed on strips for 5 min, developed and analyzed using a computing densitometer (Model 300A, Molecular Dynamics, USA).

To detect specific antigen-binding capacity, monoclonal antibodies immobilised on oxidised paper were incubated for 30 min in a solution of biotinylated IgG (60 μg/ml). After washings for 30 min (one time for 15 min and four times for 5 min), the amount of specifically bound IgG was determined as described above for constructing the calibration curve. The results obtained are summarized in Table II.

Table II: Antigen-binding activity of monoclonal antibodies against human IgG Ascite Fluid Immobilised on Oxidised Paper

According to the data obtained, monoclonal anti-IgG antibodies retained their capacity to bind antigen after immobilisation on oxidised paper. Five Φμg of immobilised antibodies were able to adsorb specifically about 3 Φμg 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 B₂ with molecular weight 370 Da. Such capacity is enough for using immunoadsrbents based on oxidised paper for preparation of diagnostic dipsticks.

Example 4 - Indirect Binding and Detection of Antibodies on Oxidised Paper

Indirect binding and detection of antibodies on the oxidised paper was accomplished as follows: oxidised paper was prepared by incubating filter paper in 100 ml of 0.01M NaI0 overnight (ON) at room temperature

(~20°C) (RT). The paper was washed twice with DW. The paper was adjusted to pH by soaking the paper with a carbonate buffer 0.1 M pH 9.2 for 10 minutes. The excess buffer was drained on kimwipe, and transferred to the

Petri dishes. A secondary anti-Rabbit antibody (Gα-R)-l O μl (2x5 μl) was applied and incubated for 1 hr at RT. Block with 0.2% Tween-20 PBS, at RT for 1 hr. Wash with PBS for 10 min. Drain paper excess buffer on kimwipe.

Apply 10 μl (2x5 μl) specific antibody (α-Thromboxane) or negative control buffer (Tris-Glycine) and incubate 1 hr., at RT. Wash 5 min. in PBS. Block with 0.2% Tween-20 PBS, at RT for 1 hr. Wash in PBS for 10 min. Transfer the paper to the container holding TxB₂-Peroxidase conjugate diluted in

Assay Buffer (1 :2.5) for 1 hr at RT using the reagents of the "Thromboxane

B₂ enzyme immunnoassay (EIA) system" produced by Amersham

International pic. Wash, 1x5 min in PBS, 3x5 min in 0.1% Tween-20 PBS

(PBST), 1x5 min in PBS.

Peroxidase reaction: Drain excess buffer of the paper on kimwipe. Add peroxidase substrate - TMB (precipitating) to the paper; use 100 μl 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.

Results:

The use of 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 shaφer reaction of the immunoassay electrode. This is demonstrated by Example 5.

Example 5 - Electrode reaction

A solution containing 0.22 ng/1 of thromboxane B₂ and the reagents of the aforesaid "Thromboxane B₂ 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. Optionally, 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. For instance, if it desired that the device should display the concentration of the target substance from the electrode potential and display its value, it is possible to operate the system with a number of solutions having known concentrations of the target substance and to memorize a table corresponding to a curve representing concentration as a function of electrode potential, viz. listing concentration values versus potential values. From such a table, the CPU can calculate the concentration of the target substance in each tested body fluid.

While electronic devices capable of carrying out the above operations of the electronic measuring means could be easily designed by skilled persons, many are available on the market in the form of chips.

The term 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. Thus the term "disposable" 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. In contrast, 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.

By "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.

Referring to the Figures, Figures 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).

Referring in particular to Figures 1 and 2, 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.

Referring in particular to Figure 3, 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. Thus, 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). Optionally, a suitable DC or AC lead (not shown) for connection to a suitable power source may be provided.

Referring in particular to Figures 4 and 5, during operation of the system ( 100), 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) is poured into the vessel (1 1) at least up to the level of openings (15) such that at least some of the fluid penetrates into the reaction chamber (12) and thus comes into contact with the oxidised paper. 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.

A second embodiment of the system (200) according to the present invention is illustrated in Figures 6 to 9. Particularly with reference to Figures 7 and 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). Thus 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. In other embodiments, 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). Thus, 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¬

established between the electrode (39) and a terminal (37) of the electronic measuring means (not shown) housed in said apparatus (40). 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, illustrated in Figure 10, comprises the same structural elements as the second embodiment, with the exception of the said slot (35), as hereinbefore described, mutatis mutandis. In the third embodiment, a deeper slot (35') is provided, enabling a porous member (44), typically in the form of a rectangular strip, to be inteφosed between the oxidised paper (38) and the reaction chamber (56). Thus, 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). It has been found that 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.

In all the embodiments, the oxidised paper is preferably, though not necessarily, filter paper such as will retain any particles having a dimension above 1 1 μm. Oxidation of the paper can be carried out by processes known in the art, e.g. by incubating with 0.0 IM solution of KI0₄ for a number of hours in the dark at room temperature.

The efficient operation of the system of the present invention is evidenced by the results illustrated in Figures. 1 1 and 12. Figure 1 1 graphically represents the current developed in a kit or system according to the invention used for the immunoassay of thromboxane B₂ 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. In 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.

Further embodiments of the system, not illustrated, may be adapted to measure other physical parameters of the tested solution, besides the concentration of a target substance. One such parameter can be the conductivity of the solution, which may provide additional useful information. 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 puφose. 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. Thus, no resterilisation of the reaction chamber is needed, preventing accidental contamination thereof or delaying the use of the apparatus for the next test, and the disposablility of the disposable part is a key feature in facilitating this. Nonetheless, in other embodiments, the disposable part may in fact be partly reusable, or fully reusable, being re-sterilisable. Of course, in such embodiments, the oxidised paper would need to be replaced between tests. Further, in other embodiments comprising fully reusable reaction chambers, these could be integrally formed within the apparatus itself in a suitable manner.

While in the foregoing description describes in detail only a few specific embodiments of the invention, it will be understood by those skilled in the art that the invention is not limited thereto and that other variations in form and details may be possible without departing from the scope and spirit of the invention herein disclosed or exceeding the scope of the claims.

Claims

Claims:

1. 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.

2. Method according to claim 1 , wherein the paper is a filter paper.

3. Method according to claim 2, wherein the filter paper is adapted to retain particles of size above 1 l μm.

4. Method according to claim 2 or claim 3, wherein said oxidised paper comprises a suitable electrode in juxtaposed arrangement therewith.

5. Method according to claim 2, wherein the chemicals required in the assay comprise an enzyme and an antibody that specifically binds to said target antigen.

6. Method according to claim 2, wherein the enzyme is an oxidase.

7. Method according to claim 8, wherein the antigen is thromboxane.

8. Use of oxidised, cellulose paper as solid phase support for immobilising at least an enzyme and an antibody in an amperometric immunoassay.

9. Use according to claim 8, wherein the paper is filter paper.

10. Use according to claim 9, wherein the filter paper is adapted to retain particles of size above 1 1 μm.

1 1. Use according to claim 8, wherein the enzyme is an oxidase.

12. Use according to 8, wherein the antigen is thromboxane.

3. 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; reaction chamber in communication with said paper; 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; at least one electrode juxtaposed with said paper; and suitable electronic measuring means operatively connected to said at least one electrode for monitoring a response of said at least one electrode to said redox reaction that occurs in the reaction chamber.

14. System according to claim 13, wherein 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.

15. System according to claim 14,wherein said paper is a filter paper.

16. System according to claim 15, wherein the filter paper is adapted to retain particles of size above 11 μm.

17. System according to claim 14, wherein said at least one electrode is juxtaposed with said oxidised paper.

18. System according to claim 17, wherein said at least one electrode is a graphite electrode.

19. System according to claim 14, wherein said electronic measurement means comprises suitable amperometric circuit means for measuring an electric potential of the said at least one electrode.

20. System according to claim 19, wherein said electronic measurement means further comprises means for digitally processing a change in said electric potential generated in response to said reaction to provide a signal representative of the immunoassay.

21. System according to claim 20, further comprising suitable memory means for storing comparison values used for enabling calculation of a desired parameter representing the result of the assay from said signal, and processing means for carrying out such calculation.

22. System according to claim 21 , wherein said parameter is target concentration data.

23. System according to claim 20, further comprising a suitable electronic display adapted for displaying said parameter.

24. System according to claim 23, wherein said display is comprised in the said non-disposable component.

25. System according to claim 17, wherein said electrode comprises an outwardly extending tip with respect to said disposable component.

26. System according to claim 25, wherein said electronic measurement means comprises 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.

7. System according to any one of claims 14 to 26, wherein said 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.

28. System according to any one of claims 14 to 26, wherein said disposable part 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.

29. System according to claim 28, wherein said selection means comprises 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.

30. System according to claim 28 or claim 29, wherein said liquid in one of said reservoirs is the test liquid comprising the target antigen.

31. System according to any one of claims 28 to 30, wherein said liquid in one of said reservoirs comprises suitable chemicals required for the particular immunoassay reaction and a redox reaction.

32. System according to any one of claims 28 to 31 , wherein said liquid in one of said reservoirs comprises an oxidase-labeled antibody-antigen conjugate, wherein the antigen is the same as the analyte of interest, and components required to create a redox system with the aforesaid oxidase.

33. System according to any one of claim 28 to 32, wherein said liquid in one of said reservoirs comprises a substrate of the enzyme immobilised on said paper.

34. System according to any one of claims 28 to 32, wherein said oxidase-labeled antibody-antigen conjugate is horseradish peroxidase-labeled antibody-antigen conjugate.

35. System according to claim 33 or 34, wherein said enzyme immobilised on said paper is GOX and said substrate thereof is glucose.

36. System according to any one of claims 28 to 32, further comprising a suitable porous member inteφosed between said paper and said reaction chamber, said porous member capable of providing fluid communication therebetween.

37. System according to claim 33, wherein the porous member comprises at least 0.5 cc of voids per 1 cc of volume.

38. System according to any one of claims 13 to 34, further comprising suitable power means.

39. System according to claim 35, wherein said power means comprises at least one electrical battery.

40. System according to claim 13, wherein the antibody immobilised in the oxidised paper is that required for the determination of TXB₂ or any one of the metabolites thereof, or any one of the metabolites of TXA₂.