WO1997020215A1

WO1997020215A1 - Detection of specific entities in a sample

Info

Publication number: WO1997020215A1
Application number: PCT/IL1996/000169
Authority: WO
Inventors: Rafi Korenstein; Yosef Rosemberg
Original assignee: Ramot University Authority For Applied Research And Industrial Development Ltd.
Priority date: 1995-11-28
Filing date: 1996-11-27
Publication date: 1997-06-05
Also published as: AU7638496A; IL116163A0

Abstract

A method for detecting the presence and optionally the level of an entity, such as an antigen, a bacterial cell, etc., is provided. The method comprises preparing a specimen, incubating it in a liquid reaction medium under conditions wherein in the presence of said entity gas is produced. The produced gas is then collected in a receptacle and the electrical conductance in the receptacle is measured. A decrease in conductance, as compared to control, then serves as indication of the presence of the entity in the assayed sample. The rate in which the conductance is decreased to a predetermined level, or the extent of decrease in conductance after a predetermined period of time may be used as an indication of the level of the entity within the assayed sample.

Description

DETECTION OF SPECIFIC ENTITIES IN A SAMPLE

FIELD OF THE INVENTION

The present invention is generally in the field of diagnostic assays and concerns a method and assay system for the diagnosis of specific entities in a medium. Entities which may be diagnosed by the method and assay system of the invention include bacteria and other cells, viruses, protein- aqueous substances, etc. The present invention allows a qualitative as well as quantitative diagnosis of said antigens.

BACKGROUND OF THE INVENTION A variety of analytical methods for diagnosis of assayed entities are currently available which allow quantitative and qualitative diagnosis of bacteria, various antigens, etc. This includes a variety of assays based on antibody-antigen interaction, such as ELISA, a variety of bacteriological assays, etc. In such assays there is usually a trade-off between rapidness in which the assay can be performed and its sensitivity. For example, sensitive bacteriological assays usually require at least twenty-four hours incubation of bacteria in a growth medium to allow the obtaining of a bacterial cup. In assays intended to detect minute quantities of antigens, there is usually a requirement for signal amplification, which either increases the time of assay or has effect on the accuracy. In various assays, it is necessary to detect a small number of cells, out of a large cell population, which display a specific antigen. For example, in order to detect cancer in its early stages, it is necessary to screen for cells displaying a certain tumor marker. At the early stages of disease, such cells are only a small fraction of the total number of cells of that kind.

As another example, in sepsis, there is to diagnose a presence of pathogenic bacteria in blood to allow immunity to begin a preventive antibiotic treatment. Here again, in the early stages of infection, the number of bacteria in a sample is relatively small and in order to accurately diagnose the infection it will be necessary to incubate a sample or wait until the infection has progressed.

U.S Patent 4,728,882 discloses an apparatus for detecting concentration of analytes, including hydrocarbons, in a liquid medium. In this apparatus use is made with capacitativc electrodes and the presence of the analyte gas bubbles are formed and displaced water from the surface electrode which decreases decapitance.

U.S. Patent 5,057,430 describes a biochemica] sensor which comprises a pair of electrodes. In the presence of an analyte, an enzyme substrate combination causes the formation of gas bubbles near the surface of the sensor which alters the dielectric properties on or near the sensor surface. This change in the dielectric properties then serves as a measure for the presence of the analyte in the medium.

Methods for determining the level of gas in a liquid medium by measurement of current-voltage relationship of current passing through the medium are known also from other publications. See for example U.S. Patent 4,358,423 and Japanese Patent Publication 84-040492.

GLOSSARY

In the text below, use will be made with some terms which were adopted for streamlining the description herein. These terms and their meanings are as follows: Assayed entity - An entity, such as cells, both living and non living, molecules or molecular complexes, etc. which are diagnosed (presence on level). These include bacteria, body cells in a sample of body fluids, viruses, proteinaceous substances, nucleic acids, lipids, complexes of molecules, etc. The assayed entities which are not living matter include such which are members of a binding couple (see below).

The assayed entity may be a biological entity or a substance as found in the assayed sample (see below). Alternatively, the assayed entity may be a reaction product of a substance in a sample, e.g. complexation of the substance to an agent. In the latter example, the assayed entity will be the complex and diagnosing its presence or its level will provide an indication of the presence or level of the substance in the original sample.

Assayed sample - a sample which is assayed for the presence therein of the assayed entity. The sample may be a body fluid sample, a carrier containing a collected assayed entity, a fluid in which a collected specimen has been suspended, a food specimen, air, etc.

The assayed sample may be an original sample or may be a result of reacting such a sample with an agent, e.g. in order to complex a substance in the sample with an agent to obtain a complex which then becomes the assayed entity.

Assay system - a system of reagents and devices used in assaying the assayed entity.

Reaction Medium - a medium into which the assayed sample is introduced for measurement of the presence and optionally concentration of the assayed entity. The reaction medium will thus comprise all reagents required for the reaction which allows such measurement.

Binding couple - two substances which are capable of specific binding to one another. Examples of binding couples include biotin-avidin, antigen- antibody, receptor-ligand, oligonuclcotide-complementary oligonucleotide, sugar- ectin, etc. In the assay of the invention the assayed entity may constitute one member of the binding couple and the other member will then form part of the assay system. The assayed entity which is a member of a binding couple may be a substance in suspension or may be an entity displayed on a membrane such as a membranous antigen or receptor, in which case, an antibody or a ligand of a receptor, respectively, will be the other member which forms part of the assay system.

Diagnosis - qualitative detection of the presence of the assayed entity in a medium, as well as quantitative determination, i.e. the determination of the level of the assayed entity in the medium.

Electrical measurement - measurement to determine conductance or resistance to electric flow, performed either by alternating current or direct current measurements.

It should however be noted that for complete understanding of the above terms and the meanings which these terms have in the context of the present invention, reference should be made to the entire description below.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide a novel, relatively rapid and accurate method and assay system for the diagnosis of an assayed entity, as defined above, in a sample.

The present invention provides a novel method for the diagnosis of the presence and optionally of the level, of a variety of entities in a medium. The entities which may be assayed in accordance with the present invention include cells such as bacteria and body cells in a body fluid sample, a variety of antigens and antigenic determinants, viruses, hormones, antibodies, molecular complexes, micelles such as cholesterol particles, etc. The method of the invention is based on measuring the level of gas production, which production occurs in the presence of the assayed entity in the medium. In accordance with the invention, produced gas is collected within a receptacle, which may for example be a capillary or a tube, while measuring electrical conductance between two locations within the receptacle. The accumulated gas produces the conductance (increases resistance) and this change in conductance thus provides a measure of the presence and optionally the level of the assayed entity in the medium.

By one preferred embodiment of the invention, to be referred to herein as the "remote receptacle embodiment" the sample is introduced into the reaction medium and formed gas which is produced outside the receptacle is collected within the receptacle, which contains, a priori, an electrically conducting liquid, e.g. an aqueous solution. The accumulated gas decreases the measured electrical conductance, and thus by measuring the electrical conductance, the level of gas in the receptacle, can be determined; and since the level of gas accumulates over a period of time depends on the level of the assayed entity in the assayed sample, the presence and optionally the level of the assayed entity in the assayed sample can thus be determined. In accordance with another preferred embodiment of the invention, to be referred to herein as the "single vesicle embodiment", use is made of vesicles or microcapsules which are loaded with a reagent which under certain conditions brings to gas production, e.g. an enzyme which can catalyze a reaction in which gas is produced. The vesicles carry on their external surface a member of a binding couple, with the assayed entity constituting the other member. In this single vesicle embodiment, the assayed entity is first immobilized on a solid support and then the vesicles are contacted with the support and in the presence of the assayed entity, the vesicles will remain immobilized thereon. Non-immobilized vesicles are then washed away and following incubation under appropriate conditions, gas production will be an indication of the presence of the assayed entity in the assayed sample. The solid support on which the assayed entity is immobilized may be the walls of the receptacle, i.e. the gas is produced and collected within the receptacle. Alternatively, the solid support is other than the wall of the receptacle and the produced gas is then collected in the receptacle, as in accordance with the remote receptacle embodiment. In accordance with another preferred embodiment of carrying out the invention, to be referred to herein as the "fvrø vesicle embodiment", two types of vesicles are used, each of which is loaded with different reagents, which once brought into proximity with one another, bring to gas produc¬ tion. Such reagents are typically two enzymes, each carried in a different vesicle or microcapsule, a first enzyme catalyzing a reaction in which gas is produced and a second enzyme catalyzing a reaction which provides conditions causing the first enzyme to enter a catalytic state in which it catalyzes gas production Each of the two types of vesicles or microcapsules carry on their external surface one member of a binding couple, the other member in both cases being the assayed entity. In the presence of the assayed entity in the medium, both vesicles will bind to the antigen and will thus come into proximity with one another and consequently gas will be produced. The reaction may be carried out within the receptacle in which case gas is produced and measured in the same place, or alternatively, produced gas may be collected in the receptacle as in accordance with the remote receptacle embodiment.

In accordance with another embodiment, to be referred to herein as the "magnet embodiment", the assay system utilizes two particles. One of the particles is a vesicle loaded with a catalytic reagent which catalyzes gas production, typically an enzyme, and the other particle is either a magnetic bead or a vesicle loaded with a magnetic entity, e.g. magnetic beads. The vesicle holding the catalytic enzyme ("catalytic particle ") and the magnetic bead or the vesicle loaded with the magnetic entity ("magnetic particle ") cany on their external surface one member of a binding couple, the other member being the assayed entity. In the presence of the assayed entity, both the catalytic particle and the magnetic particle in the assayed system will bind to the assayed entity and will therefore become complexed to one another. Then by the application of a magnetic force, the so formed complexes may be localized at a desired location and the gas production reaction may then be carried there. The complexes may be localized within the receptacle whereby gas will be produced and measured in situ. Alternatively, the particles may be concentrated in a location such that produced gas will be collected in a receptacle as in accordance with the remote receptacle embodiment.

In accordance with the remote receptacle embodiment of the invention, there is provided a method for detecting an entity in an assayed sample, comprising:

(a) preparing a specimen being said sample or a fractionation product thereof comprising said entity, if present in the sample;

(b) incubating said specimen in a liquid reaction medium under conditions wherein in the presence of said entity, gas will be produced; (c) collecting the produced gas in a receptacle; and

(d) measuring electrical conductance through the capillary or tube, decrease in conductance as compared to control, being an indication of the presence of said entity in the assayed sample.

In accordance with the single vesicle embodiment of the invention, there is provided a method for detecting the presence of an entity in an assayed medium, the entity being one of two members of a binding couple which specifically bind to one another, the method comprising:

(a) preparing a specimen being said assayed sample or a fractionation product thereof comprising said entity if present in the assayed sample; (b) reacting said specimen with a solid support such that said entity, if present in said specimen, will become immobilized on said support;

(c) contacting said support with vesicles or microcapsules loaded with a reagent which can react to produce gas, the vesicles carrying on their external surface a member of said binding couple other than said entity, whereby said vesicles will be immobilized onto said support in the presence of said entity;

(d) removing non-immobilized vesicles; (e) providing conditions permitting gas production by said reagent, collecting produced gas in a receptacle and measuring a change in electrical conductance through the receptacle, a decrease in conductance being an indication of the presence of said entity in the assayed sample. In accordance with the two vesicle embodiment of the present invention, there is provided a method for detecting the presence of an entity in an assayed sample, the entity being one of two members of a binding couple which specifically bind to one another, the method comprising:

(a) providing first and second vesicles or microcapsules; the first vesicles or microcapsules being loaded with a first reagent which can react to produce gas, the first reagent being a priori in a non-active state in which there is no gas producing reaction; the second vesicles or microcapsules being loaded with a second reagent which can react to provide conditions permitting activation of the first reagent so as to produce gas; both types of vesicles or microcapsules carrying on their external surface molecules which are all a member of said binding couple other than said entity;

(b) preparing a specimen being said assayed sample or a fractionation product thereof comprising said entity if present in the assayed sample;

(c) contacting said specimen with said first and said second vesicles or microcapsules, whereby super-complexes comprising said entity and the two vesicles or microcapsules, are formed;

(d) providing conditions in which said first reagent, once activated by said second reagent, produces gas and collecting the gas in a receptacle; and

(e) measuring electrical conductance through the receptacle, a decrease in conductance being an indication of the presence of said entity in the assayed sample.

In accordance with the magnetic embodiment of the present invention, there is provided a method for detecting the presence of an entity in an assayed sample, the entity being one of two members of a binding couple which specifically bind to one another, the method comprising:

(a) providing first and second particles; the first particle being vesicles or microcapsules loaded with a reagent which can catalyze gas production; the second particle being either a magnetic particle, or a vesicle or microcapsule loaded with a magnetic particle; the first and the second particles carrying on their external surface molecules which are all a member of said binding couple other than said entity; (b) preparing a specimen being said assayed sample or a fractionation product thereof comprising said entity if present in the assayed sample;

(c) contacting said specimen with said first and said second particles, whereby super-complexes comprising said entity and the two particles are formed; (d) drawing said super-complexes to a desired location by means of a magnetic force;

(e) providing conditions in which said reagent catalyzes gas production and collecting produced gas in a receptacle; and

(f) measuring electrical conductance through the receptacle, a decrease in conductance being an indication of the presence of said entity in the assayed sample.

Said reagent in the single vesicle embodiment, as well as said first reagent and said second reagent in the two vesicle embodiment of the invention, are preferably enzymes. Examples of gas producing enzymes are catalase which catalyzes a reaction in which gaseous oxygen, 0₂, is produced, carbonic anhydrase, which catalyzes a reaction in which carbon dioxide C0₂, is produced, and peroxidase which can catalyze a reaction in which fatty acids are oxidized by hydrogen peroxide into an aldehyde and C0₂. Reduction of electrical conductivity measured in the receptacle is an all or none indication of the presence of the analyte in the assayed sample: lack of any change of conductivity is an indication of the absence of the assayed entity from the assayed sample; a decrease in conductivity is an indication of the presence of the assayed entity in the assayed sample. In addition to the measurement of such an end point (decrease in conductiv¬ ity versus no change in conductivity) the rate of change in conductivity or the extent of change of conductivity may be used as a gauge for assessing the level of the assayed entity in the assayed sample: for example, the reaction may be permitted to continue until conductivity decreases essential¬ ly to zero and the time required therefor is measured and provides an indication for said level; alternatively, the reaction may be allowed to continue for a predetermined period of time, after which the conductivity will be measured and will provide a measure for said level.

The assayed entity may be an entity a priori present in a liquid, typically aqueous medium; or may be an entity originally present in another medium, e.g. a bacteria on a solid surface or an airborne substance, which is first sampled and then introduced into a liquid medium for assaying in accordance with the invention.

Also provided by the present invention is an assay system for the performance of the method of the invention according to either one of the above three embodiments. An assay system comprises, a receptacle, e.g. a capillary or tube electrodes for measuring current/voltage relationships (the current and voltage electrodes may be the same or different electrodes) and a device for measuring electrical conductance or resistance. An assay system in accordance with the single vesicle embodiment or with the two- vesicle embodiment comprise the vesicles or microcapsules utilized in these embodiments. The assay system may also comprise all reagents required for the assay, e.g. those providing conditions for gas production, such as those specified above and below.

The vesicles or microcapsules loaded with gas producing reagents and carrying specific members of the binding couple on their external surface, are novel entities and also form an aspect of the invention.

The invention will now be illustrated in the following description of specific embodiments and examples with occasional reference to the annexed drawings. BRIEF DESCRIP ION OF THE DRAWINGS

Fig. 1 is a schematic representation of an embodiment for diagnosing bacteria or other living cellular matter in a liquid sample;

Fig. 2 is a schematic representation of an embodiment for diagnosing bacteria or other living cellular matter present a priori on a solid substrate;

Fig. 3 is a schematic representation of another embodiment for diagnosing cells in an assayed sample;

Fig. 4 is a schematic representation of an embodiment for diagnosing an antigen in a sample; Fig. 5 is a schematic representation of another embodiment for diagnosing an antigen in a sample;

Fig. 6 is a schematic representation of another embodiment for diagnosing an antigen in a sample,

Fig. 7 shows results obtained in an experiment performed in accor- dance with the invention wherein bacteria in a sample were assayed;

Fig. 8 shows results of an experiment performed in accordance with the invention wherein the presence of bacteria on the skin was assayed;

Fig. 9 shows results of an experiment performed in accordance with the invention wherein an antigen in a sample was assayed; and Fig. 10 shows results of another experiment performed in accordance with the invention, wherein the presence of animal cells in a sample was assayed.

DESCRIPTION OF SPECIFIC EMBODIMENTS Reference is first being made to Fig. 1 which shows an embodi¬ ment for diagnosing the presence of bacteria in a sample. An assayed sample 11 is filtered through a filter 12 contained in a filtration device 13. Filter 12, has a mesh size such that cells e.g. bacteria, are entrapped on the filter. Filter 12 after filtration, with cells carried thereon marked 12' is then rolled into a cylindrical form, marked 12", and then inserted into a capillary 14. Capillary 14 is connected at each of its ends to electrodes 15 and 16 which are electrically connected to a conductance meter 17.

Cells contain the enzyme catalase and when a substrate for that enzyme is introduced (marked by a dotted arrow) into the capillary, e.g. hydrogen peroxide (H₂0₂), gaseous oxygen is produced. The production of gaseous oxygen brings to the formation of gas bubbles which increase electrical resistance, i.e. decrease electric conductance, and such an occurrence is thus an indication of the presence of bacteria in assayed medium 11. In order to have a specific assay, i.e. such intended for diagnosing specific bacteria, a solid support, e.g. microbeads, carrying bacteria-specific antibodies, may be used. After contacting the solid support with the assayed sample, e.g. by suspending microbeads carrying the bacteria-specific antibodies in the assayed medium, the solid support is separated from the medium, rinsed whereby only the specific bacteria remain immobilized thereon. Such microbeads can then be inserted into a capillary and the assay proceeds in the same manner as that described above.

Decrease in conductivity provides a qualitative indication of the presence of the assayed entity in the sample. In addition, such measure- ments allows also quantification of the level of the entity in the sample. The quantification can be based on the magnitude of conductance change after a certain period of time, on the rate of conductance change, e.g. the time it takes for the conductance to reach 0, etc.

Reference is now being made to Fig. 2, which shows another embodiment for assaying of bacteria, in accordance with the invention. The assay system of this embodiment comprises two vessels 11 and 22, connected to one another by a capillary 23. Two electrodes 23 and 24 are placed within vessels 21 and 22, respectively, the electrodes being connected to a conductivity meter 25. This assay system is suitable for measuring the presence of bacteria carried on a solid support 26, such as a cotton swab containing the assayed sample, e.g. a cotton swab brought into contact with the skin for assaying the presence of bacteria on the skin.

The carrier 26 is placed into the bottom vessel 21, and following introduction of hydrogen peroxide, similarly as above, oxygen is being produced, and is accumulated in capillary 23. Measurement of conductivity provides then an indication of the presence of bacteria on the solid sup¬ port 16. Here again, it is also possible to quantify the level of the bacteria in the sample, similarly as above.

Bacteria or cells can also be detected by allowing them to bind to the internal surface of a capillary tube. The manner of performance of the method in accordance with this embodiment is shown schematically in Fig. 3. Internal walls of a capillary tube 31 are coated with antibodies 32 which are specific for an antigen displayed on a cell to be assayed, which may be a bacteria, a certain blood cell, a malignant cell displaying a tumor marker, etc. An assayed sample 33 is slowly perfused through the capillary tube. Cells 34 which display an antigen 35 specifically recognized by antibodies 32 bind to the antibodies and remain immobilized within the capillary. The capillary is then washed from access assayed medium and then a substrate solution, e.g. a hydrogen peroxide solution is added and consequently gas is produced by the cells and gas bubbles 36 there accumulate within the capillary. The substrate solution may also, for example, be a sucrose solution and as a result of incubation of bacteria with sucrose C0₂ will be produced as a result of bacterial fermentation. Two electrodes 37 and 38, which are connected to a conductance meter 39 are inserted each at one end of the capillary. As a result of gas accumulation there will be a decrease in conductance, indicative of the presence of the specific cells in the assayed sample.

Reference is now being made to Fig. 4 showing an embodiment of the invention for detection of antigens other than cells, such as viruses, proteins, etc. This embodiment makes use of vesicles or microcapsules 41 loaded with enzymes, e.g. catalase. The vesicles or microcapsules carry antibodies 42 on their external surface. Antibody 42 is a member of an antibody-antigen binding couple. It is possible also of various applications that the vesicle will carry a member of another binding couple on their surface, e.g. a receptor where the other member will be a ligand, etc. The molecule carried on the surface of the vesicle may be a simple molecule anchored on the surface of the vesicle or microcapsule, such as antibody 42, or may be a complex such as complex 43 displayed in Fig. 4, which consists of a biotinated antibody 44 bound to an avidin molecule 45, which is in turn bound to a biotin molecule 46 carried on vesicle 41. Similarly as in the embodiment shown in Fig. 3, a sample assayed for the presence of the antigen therein is perfused through capillary 47 while providing conditions for non-specific adherence of antigen 48 to the internal walls of the capillary. Such conditions may, for example, be fixatious by treatment with alcohol followed by its evaporation; incubation of the antigen containing solution and then evaporation of this solution; coating the surface with a polycation such as polylysine; etc. Antibody 42 carried on vesicles 41 specifically recognizes and binds to antigen 48.

A reagent solution comprising vesicles 41 is then perfused to the capillary and if antigen molecules 48 are adhered to the internal walls of the capillary, vesicles or microcapsules 41 will remain immobilized within the capillary. After washing of access reagent solution, a substrate for the enzyme, e.g. hydrogen peroxide is added which results in the production of gas bubbles 49. By use of two electrodes 410 and 411, connected to conductivity meter 412, conductivity is measured and a decrease in conductivity will be an indication of the presence of antigen 48 in the assayed sample.

Fig. 5 shows an embodiment where use is made of two vesicles, each containing a different type of enzyme, in accordance with the two- vesicle embodiment. In this embodiment, one vesicle 51 is loaded with a first enzyme (El) which catalyzes a reaction where gas is produced, such as carbonic anhydrase which produces carbon dioxide, and a second vesicle 52 contains a second enzyme (E2) which catalyzes a reaction which has reaction products which cause enzyme El to become catalytically active, such as a protease which produces protons which activate the El enzyme. Both vesicles 51 and 52 carry antibodies 53 which specifically bind to antigen 54. In the absence of antigen 54, both vesicles 51 and 52 freely tumble in the solution and since their concentration is relatively low, they do not come into proximity with one another and consequently, the protons released into the vicinity of vesicles 52 do not yield activation of en¬ zyme El. Once antigen 54 is present in the solution, super complexes 55 are formed comprising antigen 54 and vesicles 51 and 52, and as a result, the protons produced by enzyme E2 reach enzyme El which then becomes catalytically active and releases carbon dioxide. Measurement of carbon dioxide directly, e.g. by collecting gas bubbles 56 in a capillary and measuring as above, or measurement of carbonate ions or measurement in pH provides an indication of the presence of antigen 54 in the assayed medium and the level of C0₂ or the extent of change in carbonate ions concentration or in the pH provides an indication of the level of antigen 54 in the assayed medium.

Reference is now being made to Fig. 6, which shows two examples of an embodiment where use is made with two particles, one being a catalytic particle 61 and the other being a magnetic particle which is either particle 62 shown in the upper part of Fig. 6 or particle 63 shown in the bottom part, catalytic particle 61 is a vesicle or microcapsule loaded with a catalytic reagent (C), e.g. an enzyme, which can catalyze a reaction in which gas is being produced. Magnetic particle 62 is a vesicle or microcap¬ sule which is loaded with magnetic particles (M), e.g. magnetic microbeads. Particles 63 are magnetic microbeads per se.

Both particles in the assay system (either particle 61 and 62 by one example, or particle 61 and particle 63, by the other example), carry on their surface antibodies 64 which specifically recognize antigen 65. In the presence of the antigen 65 in the assayed sample, super complexes, such as complex 66 comprising -particles 61 and 62 or complex 67 comprising particles 61 and 63, will be formed. Then, by the application of a magnetic force, the super complexes may be localized at a desired location where the gas production by reagents in vesicle 61 can be measured.

EXAMPLES

Example 1 - Detection of a contaminated medium A solution comprising 0.3 M sucrose, 2.25 mM Tris-HCl at pH 7.45, was exposed to air for about 12 hours at 25°C to allow it to become contaminated by bacteria. Following this exposure, the solution remained transparent showing no macroscopic signs of contamination.

In order to determine whether the solution was contaminated, 10 ml of the sucrose solution was filtered through a 0.22 μm filter, which was then introduced inside a capillary tube. Each end of the tube was connected to an electrode and the electrodes were connected to a conductivi¬ ty meter. A substrate solution consisting of 0.5 M KCI and 3% H₂0₂ was added to the capillary and the conductivity was measured. As control, a sterile solution was filtered through a similar filter, which was inserted into another capillary and measurement was performed in the same manner.

As can be seen in Fig. 6, a very clear decrease in conductance over a period of time is evident in the capillary containing the filter paper through which the assayed medium was filtered, whereas there is no change in conductivity in the control.

Example 2 - Detection of bacteria from the skin

Skin bacteria were collected by rubbing a cotton swab on the skin. The cotton swab was introduced into a flask, such as that shown schematically in Fig. 2. A clean cotton swab introduced into an identical flask served as control. After the addition of the substrate solution which was the same as that described in Example 1 , the conductivity between the two compartments was measured and the results are shown in Fig. 7. As can be seen, while the conductivity in the control did not change over a period of 10 minutes, the conductivity in the case of the assayed sample decreased rapidly, starting immediately at the beginning of the measurement.

Example 3 - Detection of cells other than bacteria

Cos 5-7 cells were allowed to adhere to internal walls of a glass capillary by incubating a suspension containing such cells within the capillary. The cells which failed to adhere to the walls of the capillary were washed out by perfusion, three times with PBS. Electrodes were then inserted into each end of the capillary as in Example 1 , and the electrodes were connected to a conductivity meter. An identical capillary to which no cells were adhered, served as control.

After addition of the substrate solution, which comprises 0.5 M KCI and 3% H₂0₂, the conductivity was measured and the results are shown in Fig. 8. As can be seen, where there is no change in conductivity in the control, there is a decrease in conductivity until 0 within about eleven minutes, in the capillary with the Cos 5-7 cells.

Example 4 - Detection of a specific antigen

A biotinated anti-mouse IgG antibody was chosen as a model antigen. In order to detect this model antigen, thylakoid vesicles were loaded with catalase and biotin was bound to the external membrane. The procedure of loading and binding was carried out as described in PCT Application having the International Publication Number WO 95/23211.

Internal walls of a capillary were covered by mouse IgG by drying a 1 .g/ml IgG solution within the capillary. The capillary was then washed three times with PBS, followed by a single wash with a solution comprising 50 μg/ml BSA (bovine serum albumin). 1.7 ng/ml of biotinated IgG was then perfused through the capillary.

The thylakoid vesicles with biotin bound thereto were incubated with Avidin and a solution comprising the vesicles was then perfused into the capillary. The vesicles were allowed to interact with the walls of the capillary for ten minutes and then the capillary was slowly washed by perfusing it with six capillary volumes of a rinsing solution comprising 0.3 M sucrose, 2.5 mM tris, pH = 7.45. Conductivity was then measured through electrodes inserted into the end of the capillary tube and the electrodes were connected to a conductivity meter. A substrate solution comprising 3% H₂0₂ was added into the capillary and the conductivity was measured. A capillary treated in the same manner but with the addition of biotin labeled IgG served as control.

The results are shown in Fig. 9. As can be seen, there is a marked decrease in conductance in the assayed capillary whereas there is practically no change in conductance in the control capillary.

Claims

CLAIMS:

1. A method for detecting an entity in an assayed sample, compris¬ ing: (a) preparing a specimen being said sample or a fractionation product thereof comprising said entity, if present in the sample;

(b) incubating said specimen in a liquid reaction medium under conditions wherein in the presence of said entity, gas will be produced;

(c) collecting the produced gas in a receptacle; and (d) measuring electrical conductance through the receptacle, decrease in conductance as compared to control, being an indication of the presence of said entity in the assayed sample.

2. A method according to Claim 1 , wherein said receptacle is a capillary or tube.

3. A method according to any one of Claims 1 or 2, wherein the gas is produced outside the receptacle and collected within the receptacle after its production.

4. A method for detecting the presence of an entity in an assayed medium, the entity being one of two members of a binding couple which specifically bind to one another, the method comprising:

(a) preparing a specimen being said assayed sample or a fractionation product thereof comprising said entity if present in the assayed sample;

(b) reacting said specimen with a solid support such that said entity, if present in said specimen, will become immobilized on said support; (c) contacting said support with vesicles or microcapsules loaded with a reagent which can react to produce gas, the vesicles carrying on their external surface a member of said binding couple other than said entity, whereby said vesicles will be immobilized onto said support in the presence of said entity; (d) removing non-immobilized vesicles;

(c) providing conditions permitting gas production by said reagent, collecting produced gas in a receptacle and measuring a change in electrical conductance through the receptacle, a decrease in conductance being an indication of the presence of said entity in the assayed sample.

5. A method according to Claim 4, wherein said solid support comprises walls of said receptacle.

6. A method according to Claim 4, wherein said solid support is placed outside the receptacle and the gas is collected within the receptacle after its production.

7. A method for detecting the presence of an entity in an assayed sample, the entity being one of two members of a binding couple which specifically bind to one another, the method comprising:

8. A method according to Claim 7, wherein step (c) is performed within the receptacle.

9. A method according to Claim 7, wherein step (c) is performed outside the receptacle and the gas which is being produced is collected within the receptacle after its production.

10. A method for detecting the presence of an entity in an assayed sample, the entity being one of two members of a binding couple which specifically bind to one another, the method comprising:

(a) providing first and second particles; the first particle being vesicles or microcapsules loaded with a reagent which can catalyze gas production; the second particle being either a magnetic particle, or a vesicle or microcapsule loaded with a magnetic particle; the first and the second particles carrying on their external surface molecules which are all a member of said binding couple other than said entity;

(b) preparing a specimen being said assayed sample or a fractionation product thereof comprising said entity if present in the assayed sample; (c) contacting said specimen with said first and said second particles, whereby super-complexes comprising said entity and the two particles are formed;

(d) drawing said super-complexes to a desired location by means of a magnetic force; (e) providing conditions in which said reagent catalyzes gas production and collecting produced gas in a receptacle; and

(f) measuring electrical conductance through the receptacle, a decrease in conductance being an indication of the level of said entity in the assayed sample.

11. A method according to Claim 10, comprising drawing said super complexes to a location so that the produced gas is collected within the receptacle after its production.

12. A method according to any one of Claims 1-11 , wherein the reagents are enzymes.

13. A method according to any one of Claims 1 -12, comprising measuring the period of time until conductance decreased to a predetermined level, this period of time being an indication of the level of said entity in the assayed sample.

14. A method according to any one of Claims 1-12, comprising measuring the decrease in conductivity over a predetermined period of time, said decrease of conductivity being a measure of the level of said entity in the assayed sample.

15. An assay system for carrying out the method of any one of Claims 1-14.

16. An assay system according to Claim 15, comprising a receptacle, electrodes for passing current and for voltage measurement and a device for measuring electrical conductance or resistance

17. An assay system according to Claim 15 or 16, comprising reagents for carrying out the method.