MXPA97008428A - Device and method for the detection of diagnost - Google Patents

Abstract

The present invention relates to an improved test cell for detecting the presence of an analyte in a liquid sample. The device has an elongated cover (10) defining a liquid sample inlet, a reservoir volume, a test volume and a window (16) through the cover in the test volume. Arranged within the cell is a sample absorber, a novel biphasic substrate and a reservoir, together capable of transporting an aqueous solution within the envelope along a flow path extending from the sample inlet through the test volume and towards the deposit volume. The invention further comprises a method for detecting the presence of an analyte in a liquid sample using the device and a biphasic chromatographic material to carry out the method. The release medium is, for example, an absorbent paper, and holds a band of, for example, metal liquid antibody-colloid, and a band of, for example, antibody-biotin, which are bonded to the first and second. epitopes respectively of the analyte. The capture site is, for example, nitrocellulose or nylon, which supports an immobilized capture component, for example, streptavidin. A second control site may be present. The release medium and the capture site are linked by overlapping to form the chromatographic substrate.

Description

DEVICE AND METHOD FOR DIAGNOSTIC DETECTION BACKGROUND OF THE INVENTION The present invention relates to analysis for an analyte, such as an antigen, in a liquid sample, such as a body fluid. More particularly, the present invention relates to a method and device for the detection of an anatous in a fluid of the body. body using a lateral flow test cell containing a novel biphasic chromatographic substrate Many types of ligand-receptor assays have been used to detect the presence of various substances in body fluids, such as urine or blood. These analyzes typically involve reactions of antigen-antibody, synthetic conjugates comprising enzymatic, fluorescent or visually observable labels and specially designed reactor chambers In most of these analyzes, there is a receptor (eg, an antibody), which is specific to the selected antigen, and means to detect the presence and / or quantity of the antigen-an reaction product The most current tests are designed to make a quantitative determination, but in many circumstances all that is required is a positive / negative indication Examples of such qualitative analyzes include blood type, pregnancy test and many types of upnalisis For these tests, visually observable indications are preferred such as the presence of agglutination or a change of color. Positive / negative analyzes must be very sensitive, due to the generally small concentration of the ligand of interest in the test fluid. False positives can be Problems, particularly with agglutination and other rapid detection methods such as dipstick and color change tests Because of these problems, sandwich analysis and other sensitive detection methods have been employed, which use liquid metal colloids or other types of colored particles However, these techniques do not They have solved all the problems encountered in these rapid detection methods. It is an object of the present invention to provide a device and method for improved detection, which have higher sensitivity and discrimination for target analytes. Another object of the invention is to provide a device of analysis, which is simpler to manufacture COMPENDIUM OF THE INVENTION The present invention provides a sensitive and rapid device and method for detecting the presence of analytes in body fluids. The method and device have a high sensitivity and result in virtually non-false positives. The use of the device and method herein provides a system of analysis which improves a minimum number of procedural steps, and reproducibly produces reliable results even when used by untrained persons. The device and the method use a single biphasic chromatographic medium, which improves the speed and sensitivity of the analysis. According to the present invention, there is provided a biphasic substrate element comprising a release means attached to a capture means located downstream of said release means. The release and capture means preferably comprise two different materials or phases having different specific characteristics The two phases are joined together to form an individual liquid path so that a front solvent can travel unimpeded from the near end (upstream) of the release medium to the far (downstream) end of the medium The release medium comprises a hydrophobic, absorbent material, such as absorbent paper. Preferred materials for use as a release medium include cotton fluff paper, cellulosic paper or paper made from cellulose together with a fibrous, polyimic material, like polyamide or rayon fibers, and a fiberglass material The primary function of the release medium is first to support and subsequently release and transport various immunological components of the assay, such as a labeled binding member and a capture component, both of which have specific affinity for the Analyte of interest This release and transport occur during the routine operation of the analysis. The capture medium comprises a polymeric, hydrophobic material, preferably a nitrocellulose or nylon membrane. The preferred materials to be used as a capture medium are microporous films or membranes, which allow the protein reagents to be immobilized directly on the membrane by passive adsorption without the need for chemical or physical fixation. For this purpose, nitrocellulose membranes, nylon 66 or similar materials are preferred, which most preferably have a pore size in the approximate scale nte 5μ to approximately 20μ The nitrocellulose membrane can be nitrocellulose alone or nitrocellulose compound ester The nitrocellulose membrane is preferably either coated or laminated onto a translucent or transparent polymeca film to provide physical support for the membrane. uses a nitrocellulose polymer, which has been cast on a polyester film such as Mylar®. Alternatively, a nitrocellulose membrane laminated on a polyester film can be used. Other backing materials can be used in addition to polyester. The primary function of the medium of capture is that of immobilizing an immunological or chemical affinity agent at one or more capture sites to capture reagents released from the release medium. As stated above, the release and capture means are joined together to form a liquid path individual. Reagents for detecting, labeling and capturing the analyte of interest are disposed on the release and capture means. Located on the release means is a reactive binding member with a first epitope of the analyte of interest. The binding member is labeled with a detectable label. A detectable component is located in the downstream release medium of the binding member, said component comprises a binding agent reactive with a second epitope of the analyte and a member of an affinity pair. The capture component is capable of forming a complex with the labeled binding member and anahto The labeled binding member and the capture component, both are releasably attached to the release means, so that when the front solvent created by the liquid sample being analyzed, passes through the release medium, the labeled binding member and the capture component both are solubilized by the liquid and flow with the solvent along the liquid path During operation, if any analyte is present in the liquid sample, it acts first with the labeled binding member, then with the captureable component as the front solvent progresses along the liquid path At the moment In which the front solvent reaches the capture means, the section of the two-phase material, the captureable complex has been formed. The capture site located in the capture means comprises the other member of the specific affinity pair for the captureable component. affinity is immobilized, preferably by simple adsorption, at the capture site, and does not progress with the front solvent. In a preferred embodiment, a control site is also located on the capture medium downstream of the capture site. The control site has immobilized, thereon, a binding agent having an affinity for the labeled binding member. The binding agent will capture any labeled binding member, which is not captured at the upstream capture site., the presence of the detectable label at the control site indicates that the sorbent transport has operated properly. The present invention also provides a device for detecting the presence of an analyte in a liquid sample. The device comprises an elongated cover housing the two-phase medium, and defines a liquid sample entry, a deposit volume, a test volume interposed between the entry and the deposit volume, and a window through the cover to observe the test result. Preferably the sample entry and the window are located on opposite sides of the roof The roof is adapted to receive the analytical materials, which are arranged on the two-phase medium sequentially inside the roof. The analytical materials comprise an additional sample absorber, the two-phase chromatographic substrate and an absorbent deposit The chromatographic medium is placed inside the cover , so that the capture site, and the control site, if applicable, are visible through the window The sample absorber, the two-phase chromatographic substrate and the tank absorber are in fluid communication and together form a path In a presently preferred embodiment, the device comprises a cover defining a sample inlet, a test volume and a reservoir volume. Arranged within the cover are a sample absorber, the two-phase chromatographic substrate and the reservoir absorber. sample absorber is disposed within the cover, opposite to the sample inlet Located downstream of the sample absorber is the biphasic chromatographic substrate comprising a release medium and a capture medium joined together to form a single liquid path. release preferably comprises absorbent paper, and the capture medium preferably comprises nitrocellulose membrane The release and capture means preferably both are laminated on a transparent plastic sheet or film Arranged on said release means is (i) a binding member comprising a specific binding protein for example an antibody monoclonal reactive with a first epitope of said analyte, said antibody being labeled with a visually detectable marker, such as colloidal gold particles, and (n) a captureable component comprising a biotinylated binding protein, eg, an antibody preferably arranged in a current below said labeled antibody The biotinylated antibody is reactive with a second epitope of the analyte and is capable of forming a complex with the labeled antibody and the analyte disposed on the capture medium is a capture site to capture and immobilize the complex. capture has immobilized on it a compose capture entity, which has a high affinity for the biotin portion of the complex, preferably streptavidin. The biphasic chromatographic medium preferably further comprises a control site disposed on the capture medium downstream of the capture site. The control site has immobilized on the same an agent capable of capturing said labeled antibody The primary function of the control site is to capture and immobilize the labeled antibody, which has not been captured at the capture site. In the currently preferred mode, the control site has immobilized on the same polyclonal antiserum specific for the labeled antibody The appearance of color of the gold particles at the control site indicates an appropriate functioning of the test, without considering the presence or absence of the analyte in the sample. control should be visible through the window of the cover In the method of the invention the near end of the two-phase substrate is in contact with the liquid sample that is being analyzed The liquid sample travels driven by the effects of the surface, such as by capillary action along the liquid path formed by the substrate. If the analyte of interest is present in the sample, it reacts with the labeled binding member and the capture component, forming the captureable complex, followed by the reaction of the complex with the capture component immobilized at the capture site This procedure results in a marked complex accumulating at the capture site The presence of the anahto is determined by observing the presence of the detectable marker at the capture site If no anahto is present In the sample, the captureable complex is not formed and no detectable marker will be present at the capture site. If a control site is present, the unbound complex or the free labeled binding member will accumulate at the control site. The invention can also be designed to exploit conventional "sandwich" or "competitive" techniques. In the case of the sandwich technique, the labeled binding member comprises an antibody, which binds to an epitope on the analyte of interest to form a complex of labeled antibody-antigen This complex then migrates to the capture site to react with a component catchable which, in this embodiment, comprises a second antibody specific for a second epitope of said analyte. For example, in the case of biotin, the affinity member may be streptavidin. At the capture site, the analyte and the labeled antibody react with the capture member immobilized to form a "sandwich" of the second antibody, analyte and labeled antibody This sandwich complex is progressively produced at the capture site as a continuously passing sample More and more labeled conjugate is immobilized at the capture site, the colored particles are added and become visible through the window of the cover, indicating the presence a of the analyte in the liquid sample Both in the presence and in the absence of a detectable level of analyte, the colored particles come together at the control site, which is also visible through the window. In the case of the technique competitively, a known amount of the target of interest is present in the release medium disposed upstream of an antibody specific for the analyte present in the release medium is labeled. The labeled analyte in the release medium may comprise, for example, an authentic sample of the analyte, or a fraction thereof, which has comparable affinity for the antibody. As the liquid sample is transported along the medium of release, the labeled analyte present in the release medium and any analyte present in the analyte. the sample compete for antibody-binding sites. If no analyte is present in the sample, the labeled analyte-antibody is added to the capture site. ura, and the presence of color indicates the absence of detectable analyte levels in the sample. If the analyte is present, the amount of labeled analyte, which binds to the test site, is reduced by the binding of the analyte in the sample. with the antibody, and no color or paler color develops. Alternatively, the system described for "sandwich" analysis can be used. The antibody specific for the analyte is biotinylated, streptavidin being immobilized at the capture site. The use of the color particle detection system in combination with the single biphasic substrate allows the construction of a family of extremely sensitive analysis systems, which minimize the occurrence of false positives and which can be effectively used by non-users. trained.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will now be more particularly described with reference to and as illustrated in, but not limited to, the accompanying drawings, in which. Figure 1A is a top plan view of a modality of a test cell useful in the device and method of the present invention showing the indicator window; Figure 1B is a longitudinal side view of the device of Figure 1A; Figure 1C is a bottom plan view of the device of Figure 1A, Figure 1D is a tail end view of the device of Figure 1A, Figure 1E is a perspective view of a currently preferred device constructed in accordance with present invention, Figure 2 is a schematic top view of the test device formed by the sample absorber, biphasic substrate and deposit material, Figure 3 is a schematic top view of a two-phase substrate of the present invention. Figure 4 is a schematic side view of the test device of Figure 2 Figure 5 is a schematic top view of the test device constructed in accordance with the invention, Figure 6 is a schematic top view of a test substrate for use in a rod mode Level of the present invention, Figure 7 is a perspective view of a dipstick mode of a test cell. useful in the device and method of the present invention In the drawings, similar reference characters in the respective Figures indicate corresponding parts DETAILED DESCRIPTION OF THE INVENTION The method of the invention involves the use of a novel biphasic chromatographic substrate to obtain an easily readable, sensitive and reproducible indication of the presence of an analyte, such as human codon gonadotropin (hCG), or luteinizing hormone (LH), in a sample of test such as, for example, a human urine sample The method and device can also be used to detect the presence of infectious agents in the blood, plasma, mucosa or other body fluid. The biphasic chromatographic substrate of the present invention forms the basis for immunologically based diagnostic tests for the detection of several analytes The use of the substrate in a diagnostic device allows the operator to determine with high accuracy and sensitivity, the presence or absence of a biological marker, which is indicative of a condition or physiological state The biphasic chromatographic substrate involves the union of two different med ios, each with a specific function The release medium has disposed thereon two diffusible reagents, dried a specific binding member to a particular site on the analyte labeled with colloidal gold or other direct mark, and a capturable component comprising a specific binding member for a different site on the analyte conjugated to an affinity pair member After reconstitution, when in contact with the test solution, and in the presence of the analyte, the diffusible reactants react with the analyte to form a diffusible sandwich, which is transported by capillary action to the capture medium. The capture medium contains two dry, non-diffusible reagents: a capture component comprising the other member of the affinity pair and a specific reagent for the labeled binding member. After diffusion in the capture medium, the diffusible sandwich is concentrated by the interaction of the capture affinity member with the capture affinity portion producing a visual signal. The biphasic chromatographic substrate comprises a release medium attached to a capture medium, such that it forms a single liquid path. The release medium is formed from a substance, which allows the release of indicator reagents, and the capture medium is formed from a substance, which allows the immobilization of reagents for detection. The release means is preferably composed of a hydrophilic absorbent material, its primary function being to maintain, release and transport various immunological parts of the test such as the labeled test component. This release and transport occur during the routine operation of the test procedure. Useful materials in the formation of the release medium include, for example, cotton fluff paper such as S & amp;S 903 and S &GB002 (available from Schleider and Scuell, Inc., Keene, NH), and BFC 180 (available from Whatman, Fairfield, NJ), and cellulosic materials such as Grade 939 made from cellulose with polyamide and Grade 1281 made of cellulose and rayon with polyamide (available from Filtertek, Inc.) and glass fiber such as Lydall borosilicate (available from Lydall, Inc. Rochester, NH). The delivery medium is preferably coated with an aqueous solution containing bovine serum albumin (BSA) and a nonionic surfactant, such as Triton X-100 (available from Rohm &Haas Co., Philadelphia, PA) with the In order to avoid non-specific binding and facilitate the release of diffusible reagents. A combination of approximately 3% BSA and approximately 0.1% Triton X-100 is useful for this purpose. The capture medium preferably comprises a microporous polymer film of nitrocellulose, nylon 66, a combination of the two, or various other materials of a similar nature, which are known to those skilled in the art. The pore size preferably is in the range of about 5μ to about 20μ. The primary function of the capture medium is to immobilize the non-diffusible reagents used to detect the presence of the analyte in the test. The protein reagents can be immobilized on the capture medium by absorption, without the need for chemical or physical modifications. The nitrocellulose may comprise nitrocellulose alone or in combination with a nitric acid ester and / or other acids. In a preferred embodiment, the nitrocellulose is directly cast on a transparent polymer film. Commercially available polyester films such as those available under the trade name Mylar® are useful for this purpose (Mylar® is a trademark of DuPont DeNemours Company). The nitrocellulose membrane can be manufactured by art-recognized techniques, including direct casting of the nitrocellulose polymer on a polyester sheet, or by laminating a nitrocellulose film with a polyester sheet The prewired or pre-cast sheets useful in the present invention are commercially available, for example, from Millipore Corporation, Bedford, MA and Corning Costar , Norpstown PA Both media are in the form of flat strips, which are joined together to form an individual flow path. In a preferred embodiment the release means and the capture means are joined by overlapping the downstream edge of the release means on the edge upstream of the capture medium then to dhirring the resulting biphasic material to a transparent polymer film or sheet, thereby keeping the media in place. The method for manufacturing the only biphasic chromatographic medium used in the present invention is described in detail in the co-pending EUA application series No [No of Proxy CWP-026] filed on the same date therewith, the description of which is incorporated herein by reference. In summary, the release means and the capture means are placed so that they overlap slightly, and an adhesive is disposed on the back of each one (the backside being the opposite side that will receive the reagents) The adhesive can be any pressure sensitive or heat fusion adhesive, which does not fill the pores of the release or capture medium, thus allowing the unimpeded flow of the front solvent through the media. The adhesives useful in the present invention are commercially available, or example, from Adhesives Research Corp. In a currently preferred embodiment, the adhesive is disposed on a transparent polymer backing The overlapping release and capture means are then passed through the lamination rolls of a lamination machine together with the backing adhesive forming a laminate of the capture and release media the adhesive and the polymer backing The resulting laminated two-phase substrate is then ready to receive the reagents which are deposited as continuous strips on the top of the substrate. the reagents have been deposited and dried, if necessary, the substrate is cut to the desired size The diffusible and non-diffusible reagents can be applied to the release and capture means, respectively, by any well known technique. In a currently preferred embodiment, the diffusible antibody reagents are applied to the release medium by direct application on the surface of the medium and dry to form a narrow band Non-diffusible reagents are applied to the capture medium by passive adsorption. For use, the two-phase chromatographic substrate is disposed within a test device said device also forms part of this invention. The device comprises, at a minimum, one housing keeping the two-phase system for conducting the analysis A preferred accommodation configuration is shown in the design request series No. 29 / 023,294, filed on May 23, 1994, which is incorporated herein by reference A particularly preferred embodiment of the cover is described in to co-pending application series No. [Non-proxy CWP-025] filed on the date thereof, which is incorporated herein by reference. The method and device of the invention cooperate to allow untrained personnel to reliably analyze a liquid sample for the presence of extremely small amounts of a particular analyte, while avoiding false positives and simplifying test procedures The invention is ideal for use in a test kit of free acquisition analysis, which will allow a consumer to self-diagnose eg pregnancy , ovulation, venereal diseases and other diseases, infection or clinical abnormality, which results in the presence of an antigenic marker substance in a body fluid, including the determination of the presence of drug metabolites or toxins. The procedure of analysis and device are specifically engineered to detect r the presence of a preselected individual analyte present in a body fluid In addition to the biphasic chromatographic substrate, the device may comprise a sample absorber disposed within the envelope near the chromatographic substrate and in fluid communication therewith. The sample absorbent preferably is a hydrophobic absorbent material, which facilitates the adsorption and transfer of a fluid sample to the biphasic chromatographic medium. Such materials may include cellulose acetate, hydrophilic polyester, and other materials having similar properties. A combination of absorbent materials Preferred materials include bound cellulose acetate, bound olefin or hydrophobic polyester, such as those commercially available from American Filtrona Company (Richmond, VA). Other preferred materials include absorbent papers such as Grade 939 or Grade 1281, available Filters of Filtertek Inc. The sample absorbent is preferably coated with a pH regulated solution containing BSA and a nonionic surfactant, such as Triton X-100. The presence of BSA and the surfactant minimize the non-specific absorption of the Analyte A concentration of about 1% BSA and about 02% surfactant in tris pH regulator is effective for this purpose. The device may further comprise a reservoir absorber disposed downstream of the chromatographic substrate and in fluid communication therewith. By providing a reservoir of absorbent material disposed beyond the chromatographic substrate, a relatively large volume of the test liquid and any analyte it contains can be expelled through the test area to aid sensitivity. The reservoir material preferably comprises a hydrophilic material , which can be equal to the sample absorber upstream. The purpose of the reservoir absorbent is to facilitate capillary action along the chromatographic substrate and to absorb excess liquid contained within the device. The reservoir absorbent preferably comprises absorbent paper made of long cotton fluff fibers, such as S &S 300, S &S 470 and S &S 900, (available from Schleider &Schuell, Inc.) or cellulosic materials, such as Grade 3MM (available from Whatman) and Grade 320 (available from Alhstrom). Broadly, the device and The method of the invention can be used to detect any analyte, which has been, so far, analyzed using known immunoassay methods, or is detectable by methods, using polyclonal or monoclonal antibodies or other proteins. Various reactive assay protocols, and analytes specific, useful in the practice of the invention, are known per se, see, for example, U.S. Patent No. 4,313,734 and U.S. Patent No. 4,366,241 The combination of aspects believed to be responsible for the excellent sensitivity and reproducibility of constructed analyzes according to the invention, it is the use of the novel biphasic chromatographic substrate and the use of a liquid colloid of metal or other color system as a marker system, which allows direct visual observation of color development. Filtering media can also be included, which limits the introduction to the test site of contaminants from the sample. The analysis is conducted simply by placing the input of the device in contact with a liquid test sample The device cover can be configured to allow direct contact with a body fluid, or as a dipstick in a body fluid container or other test solution. contact with the test fluid, one then merely expects the test sample to pass through the biphasic chromatographic substrate and into a reactive contact with the test site (and optionally one or more sites) visible through a window or windows in the outer cover of the device In a preferred embodiment the specific marking member for the ana lithium is disposed in conserved form on the release medium in the flow path within the device. If the analyte is present in the sample, it passes through the inlet and the interior of the device along the chromatographic substrate where, in the sandwich mode, reacts with the labeled binding protein, and a capture component conjugated with an affinity agent. The complex formed by the anate, the labeled binding member and the affinity conjugate then reacts with a capture affinity agent. immobilized at the capture site, which is specific for the affinity agent on the conjugate A complex is formed at the capture site comprising an immobilized capture agent-capture-analyte conjugate-labeled binding member. The presence of the complex, and thus of the analyte, is indicated by the color development caused by the aggregation of the liquid metal colloid particles at the capture site. From the foregoing, it will be apparent that the success of the method herein depends on the analyte present in the sample reacting with the labeled binding member, or on the reproducible competition between the analyte and the binding member for binding sites in the the capture site. According to the invention, as noted above, the labeled binding member and the captureable conjugate are preferably arranged in a conserved form, eg, air-dried or freeze-dried, on the release medium within the device upstream of the capture and control sites. The analyte, if any, that passes through the device and enters the liquid moves to contact the labeled binding member and the capture component forming an immune complex or initiating in situ competition as the flow continues , said complex is finally captured by the reagents immobilized on the capture means. Referring now to the drawings, Figures 1A-E schematically illustrate one embodiment of a test device 5 constructed in accordance with the invention useful for explaining its construction principles. This comprises an outer, molded cover 10, which defines an elongated, hollow enclosure. The cover 10 defines a test liquid inlet 14 and an opening 16 comprising a window through which the capture and control sites are visible. As illustrated in Figures 1A-E, the window 16 is disposed on one side of the cover 10 opposite the sample inlet 14 This configuration reduces the incidence of contamination of the test site, which is disposed within the interior of the cover 10 and is exposed through the window 16 The cover 10 further defines ventilation openings 38, 40 and 42 located along the sides and at the distal end of the cover 10 The ventilation opening 38 reduces the incidence of " vapor closure "inside the device during use The presence of openings 40 and 42 helps to reduce the" flood "of the chromatographic substrate, which can occur when the user applies too much sample to the Figure 2 illustrates schematically a preferred embodiment of the analysis materials, which when the device is fully assembled, are disposed within the cover 10 The analysis materials comprise the absorbent material 12, the two-phase chromatographic substrate 18 and the reservoir 24 The analytical materials and the interior of the cover 10 together define a flow path that generally passes from right to left in Figures 1A, B and C When the test device is placed with the inlet 14 disposed within or from another In contact with a sample of liquid, the liquid is transported by capillary action, penetration, or simple wetting along the downstream path through the absorbent 12, along the chromatographic substrate 18, and into a reservoir 24, generally as represented by the arrow The absorbent material 12 disposed inwardly of the ada 14 also serves as a filter, which can remove impure test samples, particulate matter, and interference factors. Figure 3 schematically illustrates the biphasic chromatographic substrate 18, which comprises a release medium 30 and a capture medium. Librably disposed on the release means 30 is a band of a dehydrated labeled binding member, eg, liquid metal antibody-colloid. As a liquid sample moves past the band 26, the labeled binding member enters the cell. liquid, reconstituted, and reacts or competes with any analyte present in the liquid sample. Disposed downstream of the labeled binding member is a band 28 of dehydrated capture complex. The capture complex comprises a binding member, which is attached to a second epitope of the analyte, for example, an antibody, and a captivable affinity component, for example, biotin The capturable complex it also enters the liquid sample as it proceeds along the substrate 18. Immobilized on the capture means 32 is, respectively, the capture site 34 and the control site 36. In Figure 3, the control sites and the capture are illustrated being arranged serially along the flow path. Alternatively, the site or sites of control and capture may be arranged collaterally or in other spatial relationships. The capture site 34 comprises a preselected amount of a specific capture affinity member for the catchable affinity component on the release medium. The captive component is immobilized in its place within the flow path. For example, when the captive affinity member is biotin, the capture component may be streptavidin. The control site 36 comprises immobilized antisera or antibody specific for the labeled binding member. Figure 4 schematically illustrates a side view of the operative portion of the analysis materials. As shown, the absorbent material 12 is disp in the near release means 30, and overlaps the release means 30 at one end. The release means 30 in turn overlaps the capture means 32, which is disp distantly to the release means 30. The reservoir 4 overlaps the distal end of the capture means 32 These four components together form a single fluid path, and cooperate to cause the sample liquid to flow from the absorbent 12 along the release medium 30 and the medium FIG. 5 schematically illustrates a currently preferred embodiment of the operating portion of the analysis device. As shown, the release means 30 is releasably disp thereon a band of the marked attachment member 26, which in the preferred embodiment is a monoclonal antibody conjugated to gold liquid colloid particles arranged downstream of band 26 is band 28 comprising the capture component, which in the preferred embodiment is a second monoclonal antibody specific for the same analyte conjugated to biotin. Band 28 is also releasably disp on release medium 30 Located downstream of the medium of release 30 is the capture means 32 that has immobilized thereon the capture site 34 which in the preferred embodiment is streptavidin. Located on the capture means 32 downstream of the capture site 34 is the control site 36 which in the preferred embodiment it is polyclonal antiserum specific for the labeled antibody of band 26 The invention is not limited by the precise nature of the capture site 34 and the corresponding control site 36, and in fact the control site 36 can be completely eliminated if desired Generally, the antibody or other affinity agent can be immobilized at the capture site 34 and the control site 36 using absorption, adsorption, or ionic or covalent coupling according to per se known methods. The capture medium 32 is preferably selected to bind the capture reagents without the need for chemical coupling. Nitrocelluland nylon both allow the non-chemical binding of the capture component as they control the reagent As shown in Figure 5, disp downstream of the capture means 32 is the reservoir 24 comprising a relatively large mass of absorbent or superabsorbent material The purpof the reservoir 24 is to ensure that a reasonably large amount of test liquid be extracted through the chromatographic medium. Figure 6 is a schematic illustration of one embodiment of the analytical materials useful for performing dipstick analysis. In the embodiment shown, the sample absorber is omitted. 12, and the release means 30 acts as the absorbent of mu The polymer antibody and the monoclonal antibodies or fractions thereof, which have specific binding properties and a high affinity for virtually any antigenic substance, which are useful in the present invention as binding members and capture materials, are known and are commercially available or can be produced from stable cell lines using well-known cell fusion and screening techniques The literature is replete with protocols for producing and immobilizing proteins See, for example, Laboratory Techniques in Biochemistry and Molecular Biology, Tijssen, Vol 15 Practice and Theory of Enzyme Immunoassays, Chapter 13, The Immunization of Imunoreactants on Solid Phase, pp. 297-328, and references cited therein Liquid colloids of metal and other types of color particles useful as marker substances in Immunoassay procedures are also known per se See, po Example, patent of E U A No 4,313,734 For details and engineering principles involved in the synthesis of color particle conjugates see Hopsberger, Evaluation of Colloidal Gold as a Cytochromic Marker for Transmission and Scanning Electron Microscopy, Biol Cellulaire, 36, 253-258 (1979) Leuvermg et al., "Sun Particle Immunoassay", J Immunoassay, 1 (1) 77-91 (1980) , and Frens "Controlled Nucleation for the Regulation of the Partial Size in Monodisperse Gold Suspensions", nature, Physical Science, 241 20-22 (1973) In a currently preferred embodiment, the immunoassay device of the present invention is designed to detect the human pregnancy In this embodiment, the labeled binding member is a monoclonal antibody (MAb) against human coponic gonadotropin (hCG) labeled with colloidal gold. For this purpose MAb designated 2G9 (available from Carter-Wallace, Inc.) is preferred. hCG labeled with biotin are used for the captureable complex Monoclonal antibodies, which can be used for this purpose, include hCG-specific monoclonal antibodies designated as 2B2 and B109 (available from Carter-Wallace, Inc) and CCF01 (available from Scppps Laboratory) Methods for conjugating biotin to antibodies are well known and do not form part of the present invention. In the preferred embodiment herein, the site The capture site comprises streptavidin, which has a high affinity for biotin. A control site is preferably located downstream from the capture site. The control site has immobilized on it the same goat anti-mouse IgG specific for labeled anti-hCG (available from Scantibodies Laboratory) In another preferred embodiment, the immunoassay device of the present invention is designed to detect human ovulation. In this embodiment, the labeled binding member comprises MAb 2G9 which is specific for luteinizing hormone (LH) and hCG, labeled with colloidal gold The capturable complex comprises MAb LH26 specific for LH biotini side (available from Carter-Wallace Inc) The pre-capture site It comprises streptavidin and the control site comprises goat anti-mouse IgG specific for labeled MAb. In another embodiment, the device can be adapted to detect infectious agents, such as streptococcus. In this embodiment, the labeled binding member is a polyclonal antibody of rabbit specific for streptococcus labeled with colloidal gold or other direct marker The captureable complex is the same polyclonal antibody conjugated to biotin, and the capture and control components comprise streptavidin and goat anti-rabbit IgG The cover 10 may have various shapes Typically It will comprise an elongated cover comprising interlock parts made of a plastic material such as polyvinyl chloride, polypropylene, polyester or polyethylene. Its inner flow path will contain a relatively inert material or a combination of suitable materials to facilitate the Liquid transport Cover 10 can of being adapted for direct contact with a sample liquid, as shown in the embodiment illustrated in Figures 1A-E, or it may be adapted to the shape of a dipstick, which is not shown herein, but is well known in the art A currently preferred design for the cover 10 is described in the co-pending design application series No. 29 / 023,294, filed on May 23, 1994 and in the co-pending application No. [Proxy No. CWP-025] filed on the same date with same From the foregoing it should be evident that the advantages in reproducibility, sensitivity and prevention of false positives of the analysis systems constructed in accordance with the invention, are traceable to a combination of aspects of the invention During use, the biphasic chromatographic substrate results in efficient transport of the reagents, which allows a more sensitive detection of the analyte. The present invention is now particularly described with reference to the following examples In the examples the test devices are described with reference to Figures 1-6 of the accompanying drawings, which have been briefly described before EXAMPLES Pregnancy Test The currently preferred configuration for the cover for the test device that modalizes the invention is shown in Figures 1A-E The currently preferred configuration of the test materials, including the sample absorber, the two-phase chromatographic substrate and the reservoir , shown in Figures 2-5 A modification of the test materials depicted in Figures 2-5, is shown in Figure 6 As shown in Figures 1A-E the preferred test cell of the invention comprises a pair of interlocked polymetic parts, which, when the device is assembled, define an enclosure. The cover 10 is designed to receive the biphasic chromatographic substrate and the related absorbers shown in Figures 2-5. The analytical materials are disposed within the cover 10, so that the transparent polymer backing 46 is disposed opposite to the window 16 (FIG. 1A) The results of the analysis can be read through the transparent polymer layer 46, and the presence of the layer 46 prevents contamination of the test site during use During operation, the test liquid applied through the inlet 14 it is absorbed through the sample absorber 12, and soaked along the chromatographic substrate 18, which defines the flow path, towards the reservoir volume 24 In the dipstick mode, the chromatographic substrate, shown in the Figure 6, the lacking absorbent material 12, is disposed in a cover adapted to receive it, which is shown in Figure 7. In the currently highly preferred embodiment, the absorbent 12 is bonded to the hydrophilic polyester (American Filtrone); the release medium is either S &S 903 paper or S &S GB002 paper (both Schleider &Schuell); the capture medium is a cast (or laminated) nitrocellulose membrane over polyethylene terephthalate (PET) (available from Millipore Corp.); reservoir 24 is S &S 300 paper (Schleider &Schuell). The release and capture media were laminated on 5 millimeter transparent PET pre-coated with an adhesive (available from Adhesives Research). The dimensions of the absorbent material 12 are approximately 5.0 x 1.27 x 0.25 cm on each side. The dimensions of the release medium 32 of the biphasic chromatographic substrate 18 are approximately 2.8 x 0.8 x 0.06 cm, and for the capture medium, approximately 2.5 x 0.8 x 0.018 cm. The dimensions of the reservoir absorbent pad are approximately 2.0 x 0.26 x 1.06 cm. A number of these substrates were produced and further treated to adapt them to detect pregnancy through urinalysis for the presence of human chorionic gonadotropin (hCG). The test reagents were 2G9 monoclonal antibody hCG (obtained from Carter-Wallace, Inc.) labeled with colloidal gold with a size of 15-30nm; monoclonal antibody CCF01 specific for biotinylated hCG (obtained from Scripps Laboratory); streptavidin, lyophilized material reconstituted in phosphate pH regulator at a concentration of 2 mg / ml; and goat anti-mouse IgG, solution adjusted to a concentration of 1 mg / ml in a phosphate pH regulator. The test reagents were placed on the media, as shown in Figures 3 and 5 Test Protocol Two pregnancy test samples were prepared, using two-phase chromatographic media as described above. A sample designated "Test 1" was prepared, using a nitrocellulose membrane as a capture medium, which had been laminated onto a film of polyester The other sample, designated as "Test 2", was prepared using a nitrocellular membrane as a capture medium, which was cast on a polyester backing. All other aspects of the biphasic chromatographic media were identical. The test was carried out by applying solutions containing known quantities of commercially available hCG to the near end of the chromatographic medium, and allowing test solutions to proceed through capillary action through the two-phase medium. For comparison, a commercially available pregnancy test kit (First Response ™, Carter-Wallace, Inc., New York, NY) underwent the same process. The commercial test equipment uses a single phase chromatographic medium consisting of a hydrophilic cellulosic material. The differences between the test samples and the commercial test are as shown in Table 1.

TABLE 1 Commercial Product Samples of Test Half Monophase that Biphasic comprising a chromatographic consists of medium capture material (cellulosic nitrocellulose membrane) and a medium of hydrophilic release (hydrophilic cellulosic material) Reagents Movable Reagents- Molecular Reagents- (Down to 1. Antibody - 1. Antibody marked above) hCG labeled 2. Biotinylated antibody 2. Antibody Biotinylated capture reagents 3. Streptavidin Reagents 4. Goat anti-mouse IgG capture 3. Streptavidin coupled to latex 4. Goat anti-mouse IgG latex-coupled latex Latex pearl mode Passive adsorption on the immobilization in the nitrocellulose membrane material of cellulose reagents Hydrophilic capture Reagents Direct application Direct application on the material Cells in the cellulosic hydrophilic cellulose hydrophilic material Deposit Staining paper Same stream made of an up mixed fiber of cotton lint Component Mono-phasic Bi-phasic chromatographic Absorbent of Two pads of A polyester material Sample hydrophilic cellulose acetate of 50 mm x 11.4 mm of 50 mm x 12.5 x 2.5 mm mm x 5 mm The results of the test procedure are shown in Table 2 TABLE 2 Comparison of Reaction Times for Pregnancy Tests Commercial and Improved Test 1 Test 2 Commercial hCG Level Using Using Casting (mlU) Mylar Membrane Laminated membrane Rxn / time Rxn / time Rxn / time 0 neg / 1 45 neg / 1 35 neg / 1 30 25 neg / 2 06 pos / 1 15 neg / 1 45 50 neg / 1 45 pos / 050 pos / 0 50 100 pos / 2 00 pos / 040 pos / 030 300 pos / 2 00 pos / 040 pos / 035 The pink color was clearly visible at 50 mlU of human co-genomic gonadotropin for both tests, but not for the commercial product. The results indicate that the test of the present invention can detect pregnancy in a human being so soon after a missed menstrual period In early testing stages, approximately 50 negative samples from several sources ran without false positives or even limited line cases. In contrast, at 50 mlU of hCG, the commercial pregnancy test showed a negative result. Table 3 illustrates the amounts of reagents needed for the pregnancy test (Test 2) made using the biphasic substrate of the present invention compared to the commercial test. As shown in Table 3, the tests of the present invention require significantly less reagent and (as shown in Table 2) both with sensitive or more accurate.

TABLE 3 Comparison of the Use of Reagents COMMERCIAL REAGENT TEST 2: IMPROVED PERCENTAGE OF REDUCTION Antibody 0.45 OD533 0.048 OD533 89 marked with gold 1 ug 0.1 ug 90 Biotinylated antibody 7.8 ug 0.8 ug 90 Streptavidin Table 4 shows the results of actual tests performed using the pregnancy test device herein. All the versions listed in Table 4 contain the two-phase medium and reagents described above, but differ in the configuration of the cover 10. It was surprisingly found that certain physical modifications to the cover 10, particularly the introduction of vents 38, 40 and 42 (Figures 1B, 1E), resulted in significantly higher accuracy and fewer invalid results. The difference between the different versions shown in Table 4 is as follows: Version 1 This version had long poles that were easily breakable when the components were stored in plastic bags. The devices easily peeled off when they fell off a desk.

Version 2 In this version, the posts were shortened to eliminate the break. A stopper for urine was added to prevent the urine from drifting into the release medium and moisturizing the membrane.

Version 3 This version added poles to prevent the device from separating when it fell. Additional plugs for urine were included to further prevent membrane flooding. A cross bar was added to the top of the housing to push the absorbent upstream against the membrane to assist in a simple flow and clear the gold.

Version 4 The devices of version 3 were modified manually by cutting ventilations in the device. Two long side vents and a short base ventilation were cut in the upper housing to prevent membrane flooding.

Version 4 Modified The devices of version 4 were modified by cutting a window in the upper housing similar to the urine collection area in the lower housing. This device was called a "two-sided" sample, which can be applied to either the front or the back of the device. Version 5 represents the currently preferred modality of the pregnancy test device. As shown in Table 4, all tests were completed in less than 5 minutes, without presenting invalid results.

Equivalents From the above description, a person skilled in the art can easily find out the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt it to various uses and conditions. Said modalities are intended to be included within the scope of the following claims.

Claims (1)

1. CLAIMS 1. - A method for determining the presence of a liquid sample of an analyte comprising a member of a binding pair, said method comprising: a. providing a biphasic chromatographic substrate comprising a release means comprising an absorbent paper near a near end of said substrate in liquid fluid communication with a capture medium comprising microporous or nylon nitrocellulose located downstream of the release medium and near a distant end of the substrate. wherein the release and capture means together form a liquid flow path, wherein releasably disposed on the release means, is (i) a conjugate comprising a reactive binding member of the analyte labeled with a detectable label; and (ii) a capture component, which is reactive with a second epitope of the analyte to thereby form a complex of the binding member, the analyte and the capture component; and located on the capture means is a capture site for capturing said complex, said capture site having immobilized, by adsorption, on it, a capture component having an affinity for the captureable component; b. contacting the near end with a liquid sample so that the liquid flow sample travels through sorption along said liquid path to the far end thereby inducing the sequential reaction of the analyte with the binding member and the capture component for form the complex, followed by the reaction of the complex with the immobilized capture component; and c. determine the presence of the analyte by observing the presence of the marker at the capture site. 2 - The method according to claim 1, wherein said captureable component is disposed downstream of said conjugate on the release means. 3. The method according to claim 1, wherein said analyte is human chorionic gonadotropin. 4. The method according to claim 1, wherein said analyte is luteinizing hormone. 5 - The method according to claim 1, wherein said capture material is laminated to a transparent polymer layer. 6. The method according to claim 1, wherein said detectable label is a colored particle. 7. The method according to claim 6, wherein said colored particle comprises gold liquid colloid particles. 8. The method according to claim 1, wherein said captureable component is a biotinylated binding protein. 9 -. 9 - The method according to claim 1, wherein said immobilized capture component is avidin 10 - The method according to claim 1, wherein said capture material further comprises a control site located downstream of the capture site having immobilized thereon an agent, which captures the conjugate 11 - A device for determining the presence in a liquid sample of an analyte comprising a member of a binding pair, wherein a liquid sample deposited at the near end of the device travels through sorbtion along a liquid path to a distal end of the device said device comprising a biphasic substrate comprising a release means comprising absorbent paper near the near end of the device in fluid communication with a material of capture comprising microporous or nylon nitrocellulose located downstream from the medium of ation and near the distal end of the device, wherein located on the release means of said substrate is (i) a conjugate comprising a reactive binding member of the analyte labeled with a detectable label, and (n) a capture component, which is reactive with a second epitope of the analyte to thereby form a complex of the binding member, the analyte and the capture component, and located on the capture means is a capture site to capture said complex, said capture site having immobilized, by adsorption, on it, a capture component that has an affinity for the captureable component 12 - The device according to claim 11, further comprising a control site located downstream of the capture site on the capture material, said control site having immobilized thereon an agent which captures said capture conjugate 13 - The device according to claim 11 wherein said anato is human coponic gonadotropin 14 - The device according to claim 11 wherein said analyte is luteinizing hormone 15 - The device according to claim 11 wherein said captureable component is a biotinylated antibody 16 - The device according to claim 11 wherein said compose The immobilized capture medium is avidin or an antibiotomy antibody 17 - The device according to claim 11, wherein said captivable component is disposed downstream of the conjugate on the release means 18 - The device according to claim 11, which also comprises a sheet material impervious to the analyte on one side of said capture material 19 - The device according to claim 18, comprising a cover housing said substrate and defining a window near the distal end of the device to be able to see said capture site, and a liquid sample inlet near the near end of the device, said window and inlet being arranged on opposite sides of said cover. 20. A device for determining the presence of a liquid sample of an analyte, comprising a member of a binding pair, wherein a liquid sample deposited on a near end of the device travels through capillary action along a liquid path to the far end, said device comprising: a. a biphasic substrate comprising a release means comprising absorbent paper near the near end of the device in fluid communication with a capture material comprising microporous or nylon nitrocellulose located downstream of the release means and near the distal end of the device, in where located on the release means of said substrate is: (i) a conjugate comprising a reactive binding member of the analyte labeled with a detectable label; and (ii) a captureable component located downstream of said binding member, which is reactive to a second epitope of the analyte, to thereby form a complex of the binding member, the analyte and the capture component; and located on the capture means is a capture site for capturing said complex, said capture site having immobilized, by adsorption, on it, a capture component having an affinity for the captureable component, and b a enclosing cover to said substrate, said cover defining a sample inlet located at the near end of said device upstream of (i), and a detection opening located near the distal end of said device and placed either opposite to or on the same side as the capture site 21 - The device according to claim 20 comprising a control site located downstream from the capture site, said control site having immobilized thereon an agent capable of capturing said binding member 22 - The device according to claim 21 wherein said cover further defines a control opening located downstream of the opening d and detection, said control opening is positioned opposite to the control site 23 - The device according to claim 20, wherein the detection opening is located on one side of said cover opposite the sample inlet 24 - The device according to claim 20, wherein the substrate element is in the form of a flat sheet, and wherein the binding member, the catchable component and the capture site are located on a surface of the sheet 25. - The device according to claim 20, wherein the capture material is laminated to or cast on a transparent polymeric material. 26. The device according to claim 20, further comprising a sample absorber placed upstream of the substrate element at the near end of the device. 27. The device according to claim 20, further comprising a residual absorbent located downstream of the substrate element at the distal end, the residual absorbent absorbs the liquid sample that remains after traversing the liquid path.