Directed flow diagnostic device and method

Abstract: An improved device and method for analyte assay in liquid samples, wherein a porous membrane (18) with an immobilized receptor which is capable of directly or indirectly binding to the analyte is separated from a body of absorbent material (24) capable of absorbing the liquid sample by a septum (20) capable of substantially separating the porous membrane (18) from the absorbent body (24) while substantially directing the flow of the liquid sample from the porous membrane (18) to the absorbent body (24).

Description: The present invention relates to devices and methods for analyte assay in liquid samples. More particularly, the invention relates to devices and methods utilizing immobilized specific binding receptors for analytes in liquid samples derived from biological specimens. There has long been an interest in the development of assay systems which can determine the presence or amount of specific substances in samples derived from biological specimens. Over the past two decades, immunoassays, which employ naturally occurring receptors directed to specific target substances, have provided valuable diagnostic tools for detecting substances of clinical significance. There are numerous immunoassays in the prior art in which one component of an immunological pair, e.g., an antigen or antibody, is detected or measured by using the complementary partner labelled with a tag which provides a detectible signal. In one assay technique, known as competitive binding technique, the substance to be detected competes with a labelled reagent of the same substance for a limited number of receptor sites. For example, for the detection of an unknown amount of a selected antigen in a liquid sample, a known amount of the labelled antigen is added to the sample and then contacted with receptor antibody specific for the antigen. The amount of labelled antigen which binds to the antibody is inversely proportional to the amount of the unknown antigen in the sample. In another assay, known as a sandwich assay, receptor antibody is bound to a solid surface and the selected antigen in the sample binds to that antibody. A second labelled antibody capable of binding to the bound antigen is then reacted with the antigen to form an immobilized reaction product. The label in the reaction product is detected as an indication of the presence of the antigen in the sample. For the detection or measurement of an antigen using a sandwich technique, antisera have been used for many years for both the labelled antibody and for the receptor antibody on the surface. More recently, monoclonal antibodies have been used in place of the antisera in such assay. In one such system, described in Wada, et al ., Clin. Chem., 28 (9):1986-1966 (1982), the receptor antibody was directed to one subunit of a particular antigen, hCG, while an enzyme-labelled monoclonal antibody was directed to another subunit. In this assay, the receptor antibody is immobilized on the inside of the test tube to which the sample was added. Reaction on a solid surface can be relatively slow because the contact between the immobilized reagent and the analyte in the sample is limited. The assay time has been reduced by immobilizing the receptor antibody within a porous membrane, exposing the antibody molecules in a three-dimensional matrix. In many such systems the liquid sample containing the target antigen is drawn through the membrane into an underlying absorbent material. One such system, disclosed for use in a competitive binding assay, is U.S. Patent No. 3,888,629. Other systems disclosed for use in competitive or sandwich assays include U.S. Patent Nos. 4,246,339, 4,366,241, 4,632,901. However, most such assay systems allow the deposition of particulate matter on the surface of the filter, thus obscuring the results when using, for example, a chromophoric label. In addition, these systems do not provide a means for controlling the flow rate of the liquid in the sample. The system of U.S. Patent No. 4,623,461 attempts to address these problems by forcing the liquid sample to flow transversely to the periphery of the filter by having the absorbent material associated only with the peripheral area of the filter. It is claimed that this system results in decreased deposition of particulate matter on the filter surface and improved separation of the analyte. Unfortunately, as the frontal surface area of the transverse cross-section of the membrane is quite small, the liquid will necessarily take longer to pass completely through into the absorbent. In addition, the flow through characteristics could not be altered without changing to a different membrane. Thus, it would be desirable to improve the sensitivity and separation of a membrane-bound receptor assay while controlling the fluid flow characteristics of the assay system. It would also be desirable to control the fluid flow characteristics of a system by means other than the thickness and pore size of the membrane which immobilizes the reactant. This invention provides an assay device and method for detecting an analyte in a liquid sample, particularly a sample derived from biological specimen. In general the device of the present invention includes a porous reaction membrane with an immobilized receptor which is capable of directly or indirectly binding to the target substance. The device also includes a body of absorbent material located adjacent to the porous reaction membrane which is capable of absorbing the liquid sample. Positioned between these components is a separating means or septum, typically in the form of a sheet having one or more selected ports, capable of substantially separating the porous membrane from the absorbent body while directing the flow of the liquid sample from the porous membrane to the absorbent body. In particular, a storage and reaction apparatus in accordance with the invention for use in assays for the detection and/or determination of a bindable target substance in a liquid sample suspected of containing such substance comprises: (a) a liquid-permeable, porous reaction membrane having an upper and lower surface, at least one defined region of said membrane or at least said upper surface thereof having immobilized thereon a receptor capable of directly or indirectly binding to the bindable target substance; (b) a body of absorbent material capable of absorbing a liquid, said body having a surface located adjacent to the lower surface of the porous reaction membrane; (c) separating means between the said lower surface of the porous reaction membrane and the said upper surface of the absorbent material body for substantially isolating liquid flow therebetween and (d) port means through said separating means for substantially directing the flow of liquid from the lower surface of said porous reaction membrane through said separating means; whereby liquid sample applied to the upper surface of said porous reaction membrane will permeate in a selected flow pattern to its lower surface and be substantially directed to at least one selected portion of the surface of said body of absorbent material. The liquid sample is assayed to form a detectible reaction product on the membrane. In preferred assay embodiments, the liquid sample is biologically derived (e.g., urine or serum) and is suspected to include as the target substance, typically an antigen, antibody or hapten capable of being bound by the receptor immobilized on the membrane. In the detection of a selected antigen in a sandwich assay, a receptor antibody reagent (to which the antigen binds) is immobilized on the membrane. Target antigen in a fluid sample is bound to the receptor antibody. A second reagent, soluble labelled antibody capable of binding the target antigen, then reacts with the bound antigen to form a detectible reaction product on the membrane. These reactions can be simultaneous or sequential. In a preferred embodiment, the device comprises a separating means which is a liquid impermeable membrane septum or sheet substantially separating the porous membrane from the absorbent body. This septum will be provided with channels or ports located so as to direct the liquid flow preferentially toward or away from the center of the porous membrane. In another preferred embodiment, the septum comprises a body of porous material in at least one of the channels of the impermeable membrane septum. This body will provide a channel or conduit to enhance liquid flow between the porous membrane and the absorbent body only at the points of contact. In a further preferred embodiment, the receptor reagent is concentrated in at least one defined region, such as a dot on the membrane, and the detectible reaction product is formed at the dot. For example, referring to a sandwich assay for a selected antigen, the receptor antibody is immobilized at a dot on the membrane to bind the antigen. The bound antigen then reacts in turn with a labelled antibody capable of binding to the bound antigen. Multiple dots using antibodies (or antigens) specifically reactive with different antigens (or antibodies) may also be employed to simultaneously assay different antigens in the sample. In a still further preferred embodiment, the receptor reagent is immobilized on a porous membrane comprising nitrocellulose backed with paper or other material such as polyester or fiberglass. In a second aspect the invention provides an assay method for the detection and/or determination of a bindable target substance in a liquid sample suspected of containing such substances, employing as an assay device an apparatus comprising: (a) a liquid-permeable, porous reaction membrane having an upper and lower surface, at least one defined region of said membrane or at least said upper surface thereof having immobilized thereon a receptor capable of directly or indirectly binding to the bindable target substance; (b) a body of absorbent material capable of absorbing a liquid, said body having a surface located adjacent to the lower surface of the porous reaction membrane; (c) separating means between the said lower surface of the porous reaction membrane and the said upper surface of the absorbent material body for substantially isolating liquid flow therebetween and (d) port means through said separating means for substantially directing the flow of liquid from the lower surface of said porous reaction membrane through said separating means; the method comprising applying the sample, visible labelled substance, and any additional reagents to the upper surface of said permeable reaction membrane whereby the applied liquid will permeate through said port means from the membrane lower surface and be substantially directed to at least one selected portion of the adjacent surface of said absorbent material body and thereafter detecting said labelled substance bound to said upper surface as an indication of the presence or absence of target substance bound to said bindable substance on said upper surface. The invention will now be described by way of example with reference to the accompanying drawings in which: Figure 1 is a perspective view of an assay device according to the present invention; Figure 2 is a cross-sectional view, with portions broken away, of an assay device according to the present invention; Figures 3A, 3B and 3C are plan views of alternative embodiments of a septum of the device of Figure 2; Figure 4 is a cross-sectional view, with portions broken away, of another assay device according to the present invention; Figures 5A, 5B and 5C are alternative embodiments of septums; Figures 6, 7, 8 and 9 are cross-sectional views, with portions broken away, of assay devices according to the present invention. This invention provides a novel and improved assay device and method for detecting an analyte or target substance in a fluid sample, particularly a sample derived from a biological specimen. The device of the present invention includes a porous reaction membrane with an immobilized receptor which is capable of directly or indirectly binding to the target substance. The device also includes a body of absorbent material located adjacent to the porous membrane and capable of absorbing the liquid sample. Positioned between these components is separating means in the form of a septum or sheet capable of substantially separating the porous membrane from the absorbent body while substantially directing the flow of the liquid sample for the porous membrane to the absorbent body through ports or channels through the septum. Referring now to the drawings, Figure 1 depicts a representative assay device 10 according to the present invention. While the device is provided with a rectangular shape, any other appropriate shape may be employed and provided with an opening means for receiving and temporarily storing liquid which can conveniently be in the shape of an inverted frustoconical well bounded by a wall. The device of the present invention is disclosed in greater detail in Figure 2, which depicts a vertical cross-section of the device of Figure 1. As depicted therein, the device is illustrated in the form of container means defining a hollow receiving space or chamber 26. The boundaries of this space are provided herein by two components, 14 and 12, referred to herein for convenience as the base and cover, respectively. These components are suitably formed of a disposable plastic material such as polycarbonate polyethylene, polypropylene, polystyrene or polyvinyl chloride, or of a durable material such as a metal (e.g., aluminum) which permits reuse of device 10, if desired. The receiving space 26 is shown to contain a body of absorbent material 24 separated from porous reaction membrane means 18 by a septum 20, whose functions are described in greater detail below. As shown, cover 12 includes well 16 bounded by a liquid retaining ring or wall conveniently shaped to receive the liquid sample and facilitate its contact with the membrane 18. By selectively matching the size of well 16 with receiving space 26 and absorbent body 24, the operation of the present apparatus can be simplified so that, for example, the entire liquid sample or optional wash liquid can be placed in well 16 as a single step of the operation. As illustrated, membrane means 18 includes a porous reaction membrane with an externally visible upper surface. The reaction membrane may be of any type capable of immobilizing reaction product of the reagents and sample component without adversely affecting the reaction, and permits passage of the remainder of the liquid sample or of a washing solution. Suitable membranes may be formed of any material capable of immobilizing the receptor reagent employed in the practice of the present invention, such as nylon, glass fibers, or other natural or synthetic materials which can be coupled directly or indirectly to the selected receptor. The porosity of the membrane preferably varies from about 0.2 to about 12 microns. However, as a presently preferred embodiment, membrane means 18 comprises paper-backed nitrocellulose, or other types of nitrocellulose membranes with similar characteristics. This embodiment comprises a nitrocellulose membrane backed with porous paper similar to filter paper. A representative example is commercially available under the tradename BAC-T-KOTE by Schleicher and Schuell. This preferred membrane is substantially more durable than nitrocellulose alone and can be employed without any other support component. Also, it provides enhanced sensitivityto the reaction. Also, polyester supported nitrocellulose may be used such as supplied under the name NITROPLUS by Micron Separation, Inc. As described below, in a typical system, a binding reagent is first immobilized on the membrane. The reagent reacts with and captures the predetermined target substance of the liquid sample to be assayed. Such reagent, typically an immunological protein such as an antibody or antigen, can be immobilized directly or indirectly onto such membranes, such as nitrocellulose, by either adsorption or by covalent bonding. The depth or thickness of the membrane is selected so that an adequate amount of binding reagent can be immobilized to capture the sample component. However, the thickness should not be so great as to cause undue delay of the passage of the liquid sample through the membrane. The absorbent body 24 of the present device can employ any of the known and conventionally employed absorbent materials which serve to draw liquid through a porous membrane, such as, for example, by capillary action. Useful known materials include cellulose acetate fibers, polyester, polyolefin or other such materials. It has also been found convenient to use layers of commercial available filter paper, or even toilet paper, which can be selected to provide sufficient volume to absorb the liquid employed during the assay of the present invention. As seen in Figure 2, substantially isolating membrane means 18 from absorbent body 24 is separating means in the form of septum 20. Septum 20 will have outer dimensions appropriate for the container employed in the device of the present invention. For example, septum 20 as shown in Figure 3A, can be provided comprising a membrane of sufficient size so that it is retained in a position and is sealed by the opposition of cover 12 to base 14 in Figure 2. In this manner, a seal is formed which prevents liquid flow around reaction membrane 18 and channels flow through the openings or ports 22 in septum 20. Suitable septum membranes may be formed of any material which is substantially impermeable to liquid and, desirably, is not reactive with or harmed by the reagents employed in the practice of the present invention, such as membranes of plastic material such as polyethylene, polypropylene, polystyrene, polyvinyl chloride, or the like. Referring further to Figure 3A, septum impermeable membrane 20 will have located thereon a zone 21 whose boundary is an imaginary line which approximates the lower dimension of frustoconical well 16. This is the zone through which liquid would tend to flow from the lower surface of porous membrane 18 to absorbent body 24. Located within or toward the center of zone 21 in the septum embodiment of Figure 3A are passages or ports 22a, which are openings which are filled with absorbent permeable material through which liquid may flow. In a particular embodiment of the present invention, as illustrated in Figures 3A and 3C, these passages 22a and 22c, respectively, are radially spaced from the center of the immobilized receptor or typically in the center of zone 21 to direct the flow of liquid away from the immobilized receptor center and towards the periphery. As an alternative, in Figure 3B the device is provided with a passage 22b proximal to the center of the immobilized receptor which will tend to direct the liquid flow toward the center of zone 21. This phenomenon will be described more fully below. Figures 4 and 5A-C illustrate alternative septum means 20 using various combinations of an annular ring 23 of absorbent material, which can be the same or a different material as absorbent body 24. This annular ring 23 of Figure 4 provides the only separation between membrane 18 and body 24 and permits selected liquid flow contact between limited portions of the facing surfaces of the absorbent body. The ring is retained in place by pressure between membrane 18 and body 24. In effect, the septum or separation means is the open area around and within ring 23 which serves as the port through the open septum. Figure 5A illustrates a top view of ring 23a in the form used in the device of Figure 4. The outer dimension of annular ring 23a will generally approximate the boundary dimension of zone 21 or less. In this embodiment, liquid flow through porous membrane 18 will tend to be directed away from the center of zone 21 and through the passage provided by the body of ring 23a. Turning to Figure 5B, as an alternative embodiment, septum 20 is in the form of ring 23b which includes an internal portion or disk 25b of substantially liquid-impermeable material, which can be the same as the sheet of septum 20 described above. Disk 25b would tend to enhance the flow direction capabilities of annular ring 23b by preventing inadvertent contact between center portions of membrane means 18 and absorbent body 24, thus preventing resultant undirected liquid flow. Here, the separating means is the combination of the open area outside ring 23b and disk 25b. As a further alternative embodiment, septum 20 is shown in Figure 5C as a membrane of liquid impermeable material similar to that provided in the embodiment of Figure 3A. Here, absorbent material is inserted into impermeable septum sheet 20 and retains impermeable disk 25c. This complete septum provides further control over the liquid flow in the device of the present invention. Referring to Figure 6, another embodiment of the device includes a unit or device housing or boundary 14 which defines a relatively wide rim to funnel the liquid. Also, membrane 18 and absorbent body 24 extend across the entire interior of the open space leaving no annular opening as in the embodiment of Figure 4. The septum 20 is of the same type illustrated in Figure 5C. Here, as illustrated by the arrows, the liquid flows through reaction membrane 18 preferentially away from the center of the immobilized receptor, typically located at the center of the upper surface of membrane 18 exposed to the liquid. This is caused by impermeable septum 20 surrounding ring of absorbent material 20b which, in turn, retains impermeable disk 20c in its center. Thus, the liquid on the upper surface of membrane 18 can only flow through ports 20b resulting in flow away from the center of the immobilized receptor. The device of Figure 7 is similar to that of Figure 6 except for the septum which is similar to that of Figure 4. Here, the septum comprises ring 23a with a total open area to the center and outside of the ring. Effective fluid contact between membrane 18 and absorbent material 24 is made via ring 23a through which the liquid flows as in the pattern of the device of Figure 6. Like Figure 4, this illustrates that an open space can, in effect, constitute the separating means if the liquid has a path through an absorbent material port which is far less resistant to flow. Referring to Figure 8, a device is illustrated similar to that of Figure 6 but with certain important differences. Membrane 18 is in the form of a disk of a smaller diameter than that of membrane 18 in Figure 7. The most significant difference is that septum 20 is designed to cause the liquid to flow towards the center of the immobilized receptor (or center of membrane 18) rather than away from it. This is accomplished by forming port 20a in an area directly below the immobilized receptor center. In the illustrated embodiment, port 20a is a round disk shape with a diameter substantially less than that of the perimeter of the exposed upper surface of membrane 18. Here port 20a is open but, as with the other embodiments, absorbent material can be placed in the opening to serve as the port. Another embodiment of the invention is illustrated in Figure 9. As with the other devices, here the body of absorbent material 24 is pliable. The device further includes platform means, suitably in the form of pedestal or plug 30, which projects upwardly into the absorbent material towards the center of the immobilized receptor. As illustrated, plug 30 preferably is of sufficient height so that, when pressed against the absorbent material body 24, sufficient upward pressure is applied against the absorbent material to cause flexing of the flexible impermeable sheet 20, into a slightly convex configuration and to correspondingly modify the unflexed shape of the port means toward the center. In addition, where the absorbent material comprises multiple layers of fibers 32 such as filter paper or paper towels, the layers above plug 30 tend to loosely fall away like butterfly wings. In this manner, space is created between the filter paper in the form of channels, providing improved flow in comparison to tightly compressed absorbent paper. An important feature of the invention is the ability to optimize the kinetics of the reaction by directing flow of the tested sample either away from the center of the immobilized receptor or towards it by a selection of the location and type of ports through the septum. The flow rate is adjusted to be fast for convenience of the user, but slow enough that the target substance will be captured. Also, it is desirable to flow away from the center for certain high viscosity samples, such as serum, which could otherwise tend to clog the reaction membrane. Conversely, for low viscosity sample, such as urine, where clogging is not a problem and where there may be a low concentration of target substance, it is desirable to flow the liquid towards the center of the reaction membrane. In operation, the assay device of the present system broadly is used to determine the presence of analyte in a liquid sample, employing at least one reagent to form a detectible product on membrane 18 as an indication that the analyte is present in the sample. The liquid sample is contacted on one side of membrane means 18 by introduction through the opening in well 16. After permeating membrane 18, the liquid is drawn therethrough by and into absorbent body 24. However, due to the operation of the septum 20 of the present device, the flow rate and direction of this liquid can be controlled, as the liquid flow between lower surface of membrane 18 and the surface of absorbent body 24 is constrained and directed either through passages 22 or the conduit provided by absorbent member 23. The operation of assays in accordance with the present invention will be described in further detail below. For the specific devices of Figures 2-7 and 9, the flow is directed away from the center of the immobilized receptor. In a preferred embodiment, the receptor is located in a small region (e.g. a dot) of the total available upper surface of membrane 18. Here, it is preferable that the sample flow primarily in a pattern proximal to the outer boundaries of this small region. In this manner, most (preferably substantially all) of the liquid sample contacts the sample perimeter rather than being concentrated at the center of the region as it would be if the port through the septum was an open center. The latter form of port can lead to clogging of the center for a viscous fluid such as blood serum. On the other hand, the port(s) are formed so that substantially all of the sample contacts the receptor region rather than a major portion bypassing it around the region's periphery as it would if no septum were used at all. For this purpose a preferable form of the septum port is a continuous or discontinuous ring, or their equivalent that produces this annular flow. By this adjustment of the flow pattern, the sensitivity of the system for low concentration samples (e.g. HIV antibody in serum) is increased without clogging of the membrane. For the device of Figure 8, flow is directed toward the center of the immobilized receptor, e.g. in the form of a dot, by use of an open center septum. Preferably, the open center has an outer perimeter dimensioned to correspond to the outer perimeter of the dot. This embodiment is particularly useful to increase the kinetics and sensitivity for samples of low viscosity and having a target substance at a lower concentration. The system is particularly applicable to an immunoassay wherein the sample component is one component of an immunological pair including antigens, antibodies, or haptens. The immunological pair includes two components which immunologically bind to each other. Specific immunological pairs include antigens and their antibodies (monoclonal antibodies or polyclonal antibodies), biologically functional haptens and their antibodies, enzymes and their substrates, hormones and their receptors, vitamins and their receptors, biotin and either avidin or an antibody to biotin, and lectin and its specific binding mono, di- or trisaccharide. For simplicity of description, the system will be described with respect to immunoassays using the antigen-antibody immunological pair. The liquid sample is biologically derived, (e.g., urine or serum) and the one reagent comprises a labelled antigen or antibody. Referring first to a sandwich assay for the detection of antigen, the one reagent is a labelled antibody specific for the predetermined antigen in the sample. This system uses a second reagent, a capture antibody also specific for the predetermined antigen, immobilized on membrane disc 24a prior to addition of the liquid sample to the device. The techniques of immobilizing proteins such as monoclonal or polyclonal antibodies to solid surfaces such as membrane disc 24a without deactivation are well known. See e.g., Schuurs U.S. 3,551,555 and Hendry et al., J. Immun. Methods, 35 1980, 285. For plastic materials such as nylon, such proteins may be immobilized by covalent bonding, e.g., as described in U.S. 3,720,760. The amount of protein immobilized per unit area of nylon is greater than that for nitrocellulose. The labelled antibody or antigen described with respect to the sandwich assay may be any of the conventional types including radioactive, enzyme, or a metal complex label which are conjugated to the antibody. Formation of conjugates between such immunological substances and labels are well known, e.g., (a) radioactive labels - U.S. 3,646,346, Hunter et al., Nature 142 (1962), 945, (b) enzyme labels - U.S. 3,654,090, 3,791,931 and 3,817,838, Wilson et al., Immunoflourescense and Related Staining Techniques, Knapp., W. et al., Eds. L. Sevier-North Holland, Bio-Medical Press, New York-Amsterdam, 1978, pp. 215-224; (c) fluorescent quencher labels - U.S. 3,996,345; (d) radioactive labels - U.S. 4,062,733; (e) fluorescent or enzyme labels U.S. 4,067,959; (f) chemiluminescent labels - U.S. 4,104,029; (g) non-enzymatic catalyst label - U.S. 4,160,645; (h) enzyme pair labels - U.S. 4,233,402, chemically induced fluorescent labels - 4,720,450; and (i) enzyme non-ionic charge labels - U.S. 4,287,300. In addition, the labels disclosed in U.S. 4,366,241 may be employed. Also, colloidal gold labels are discussed in detail hereinafter. Colloidal gold conjugates useful for probes such as cytochemical markers are well known for microscopy. See, e.g., Scanning Electron Microscopy , 1981, II, pp. 9-31, "Immunocytochemistry" Eds. Polak, J.N., et al., Bristol, London, Boston (1982) pp. 82-112, and Journal of Neuroscience Methods , 7(1983), pp.1-18. Colloidal gold particle markers are simple to use in comparison to other conventional markers. For example, they do not require instruments necessary for detection of other markers such as radioactive isotopes. Furthermore, unlike enzymes, they do not require the additional step of adding substrate. However, they have not been used extensively for commercial immunoassay kits, perhaps because of their low level of visibility using conventional techniques for mixing reactants. For example, the sensitivity of an assay using colloidal gold conjugates in a previous membrane system has been thought to be insufficient to provide the desired level of sensitivity. It has been found that by directing the flow of the liquid sample and reagents through the membrane, the sensitivity is so improved that colloidal gold particle conjugates are useful reagents for immunoassay kits without the need to use expensive microscopes. If the sensitivity of the gold-immunological reagent conjugate is insufficient, even with the increase of the present system, a technique for enhancing the sensitivity of the gold complex may be employed such as disclosed in Holgate, C.S., et al., J. Histochem. Cytochem 31:938 (1983) and in Dancher, G, et al., J. Histochem. Cytochem 31:1394 (1983). This system is an "indirect" technique employing an immunological reagent, immunoglobulin, absorbed to colloidal gold. The gold particles are revealed by a silver precipitation reaction. In essence, the silver enhancement takes advantage of the catalytic effect of gold to catalyze the photographic physical developer process converting silver ion to silver metal. Suitable colloidal gold or gold sol particle size is from 3nm to 150nm. This immuno gold-silver staining method may have an enhanced sensitivity of up to 200-fold in comparison to the use of the gold particles without silver staining. The effect of directed flow is particularly apparent when the immunological reagent is concentrated at a specific region, such as a dot on the membrane, as described below. In that instance, the immobilized antibody is only deposited at that one dot. In conventional techniques, only the antigen in the sample in an imaginary column above the dot contacts the antibody. The remaining antigen in the sample passes through the membrane without such contact. In contrast, directing the flow of liquid sample containing the antigen causes exposure of significantly more antigen to the immobilized antibody on the membrane with the above advantageous results. This principle of increased effeciency applies to any of the conventional immunoassays with respect to which the present system may be employed. Also, the increased efficiency or interaction increases the local concentration of reactants which, in turn, increases the reaction kinetics. The present invention is also applicable to the competitive binding technique. In such system for the detection of antigen in a liquid sample, the corresponding member of the immunological pair, i.e., antibody is immobilized on to the membrane surface. Antigen labelled in the manner described above of the same immunological character as the antigen analyte to be detected in the sample is contacted with the immobilized antibody on the membrane. The immobilized antibody is in limited supply, and so a competition is set up between the antigen in the sample and the labelled antigen. Thus, the signal emitted from the label is inversely proportional to the amount of antigen in the sample. As with the sandwich assay, the competitive binding assay may be performed by reversing the roles of the antigen and antibody. In this instance, the immobilized member of the immunological pair is the antigen for the detection of antibody in the sample which competes with labelled antibody. The immunoassays which have been described are the sandwich assay and the competitive binding assay. It should be understood that the system is also useful for other immunoassays such as, for example, described in U.S. Patent No. 4,366,241. The substances to be analyzed include a wide variety of biologically derived substances, e.g., proteins. The following is a list of some of these substances. (The listed substances also include immunologically reactive antibodies, and fractions of the substances). Immunoglobulins IgE IgA IgM IgD Microorganisms Aerobacter aerogenes Aerobic Spore-Forming Bacilli Bacillus anthracis Bacillus substilis Bacillus cereus Anaerobic Spore-forming Bacilli Clostridium botulinum Clostridium tetani Clostridium perfringens Brucellae Brucella melitensis Brucella abortus Brucella suis Chlamydia (unclassifiable parasites bacterial/viral) Chlamydia agents (naming uncertain)) Chlamydia trachomatis Corynebacteria Corynebacterium diptheriae Escherichia coli Fungi Cryptococcus neoformans Histoplasma capsulatum Coccidioides immitis Candida albicans Mucor corymbifer (absidia corymbifera) Hemophilus-Pordetella group Hemophilus influenzae H. ducreyi H. hemophilus H. aegypticus H. parainfluenzae Keibsiella pneumoniae Mycobacteria Mycobacterium tuberculosis hominis Mycobacterium bovis Mycobacterium avium Mycobacterium leprae Mycobacterium paratuberculosis Mycoplasmas Mycoplasma pneumoniae Mycoplasma hominis Neisseriae Neisseria meningitidis Neisseria gonorrheae Other Pathogens Listeria monocytogens Pasteurellae Pasteurella pestis Pasteurella multocida Pneumococci Diplococcus pneumoniae Pseudomonas aeruginosa Rickettsiae (bacteria-like parasites) Rickettsia prowazekii Rickettsia mooseri Rickettsia rickettsii Rickettsia conori Rickettsia australis Rickettsia tsutsugamushi Rickettsia burnetii Salmonella choleraesus Salmonella typhimurtum Salmonella typhosa Shigella arabinotardo Shigella boydii Shigella dysenteriae Shigella flexneri Shigella schmitzii Shigella Sonnei Staphylococci Staphylococcus aureus Staphylococcus albus Streptococci Streptococcus pyogenes Groups B, C, D, F, G The Spirochetes Treponema pallidum Borrelia recurrentis Leptospira icterohemorrhagiae Leptospira canicola Toxoplasma gondii Peptide and Protein Hormones Corticotropin (ACTH) (adrenocorticotropic hormone) Follicle-stimulating hormone Luteinizing hormone (interstitial cell-stimulating hormone) Parathyroid hormone Prolactin Chorionic Gonadotropin Insulin Glucagon Relaxin Somatropin Triiodothyronine Thyrocalcitonin Thyroxine Tissue Hormones Angiotensin I and II Bradykinin Gastrin Human placental lactogen Secretin Peptide Hormones Oxytocin Vasopressin Viruses Adinoviruses Arboviruses Eastern Equine Eucephalitis Virus Western Equine Eucephalitis Virus Sindbis Virus Semliki Forest Virus St. Louis Encephalitis Virus California Encephalitis Virus Colorado Tick Fever Virus Yellow Fever Virus Dengue Virus Hepatitis Hepatitis A Virus Hepatitis B Virus Herpes Viruses Herpes simplex, Types I and II Varicella (Chicken pox) Cytomegalovirus Myxoviruses Influenza (A, B, and C) Parainfluenza (1-4) Mumps virus Newcastle Disease Virus Measles Virus Canine Distemper Virus Respiratory Syncytial Virus Rubella Virus Picornaviruses Poliovirus Coxsackievirus Echoviruses Rhinoviruses Pox Viruses Vaccinia Molluscum contagiosum The system is also applicable to other assay systems which are not categorized as immunoassays, e.g., the detection of unknown DNA sequences. For example, liquid sample containing the unknown DNA sequence is passed through the membrane and immobilize on the membrane as by contact with DNA previously immobilized on the membrane. Then, a labelled DNA probe passed in a liquid through the membrane. If hybridization occurs, the labelled DNA probe will be retained in detectible form on the membrane surface. This system is described in Polsky-Cynkin, R., et al., Clin. Chem. 31/9, 1438 (1985). As mentioned above, in a particularly effective assay, the immunological reagent is concentrated on at least one defined region on the membrane which appears as a dot but which actually extends through the membrane in a column of a diameter approximately equal to the dot. Referring to a sandwich or competitive binding techniques, this is accomplished by immobilizing the capture immunological reagent, e.g., antigen or antibody, only in such region by flowing the reagent to be immobilized through the membrane thickness. The reaction with the sample component and with the labelled reagent only occurs in that region. The dot approach has certain advantages such as the performance of multiple simultaneous assays with a single device as described below. Also it provides a more distinctive end product signal since it is concentrated at a single region. Another advantage of the dot approach is that it permits the simultaneous detection of multiple components in a sample. For example, with a single assay device, two different antibodies specific for predetermined different antigens can be immobilized on distinct spots on the membrane. The sample suspected of containing either one of the antigens is then contacted with the membrane and with labelled antibody specific for the two different antigens. A signal produced by the labels at one or the other of the dots indicates the presence or absence of one or both of the antigens. The dots may be distinguished from each other by their location or by an identification near each immobilized antibody dot. Thus, for example, if a color appears at the first dot but not the second, the first antigen but not the second antigen is present. If both dots appear, then both antigens are present, and if no dots appear, neither antigen is present. Alternatively, the label may be selected to produce different colors at each of the dots. This system could be expanded to include the simultaneous detection of more than two components of the liquid sample by a corresponding number of immobilized immunological reagent on the membrane. In some instances, a first antibody is reactive with a particular subunit of a number of different antibodies. If a second antibody is specific for a subunit of one antigen only, such second antibody can be used as the immobilized antibody and a single labelled first antibody can be used as the universal labelled antibody for antigen of interest. The standard protocols for the conventional immunoassays may be used in the present invention. For example, in a sandwich assay, the order of addition of the sample and labelled reagents may be simultaneous or sequential. While monoclonal antibodies have known advantages and purity over polyclonal antibodies, either type of immunological reagent can be used in accordance with the present invention. While the above system is described in terms of yes-no quantitative test, it should be understood that it is also suitable as semi-quantitative test using appropriate signals, such as colors, produced at different known concentrations of the component to be analyzed. Thus, for example, for the analysis of an antigen by a sandwich technique, the system can be run at progressive dilution to obtain an approximation of the color expected for a particular dilution. The unknown concentration of an antigen is compared to these colors to give an approximation of the concentration of antigen present in the sample. Further disclosure of the nature of the present invention is provided by the following specific examples. It should be understood that the examples disclosed are not intended to limit the scope of the invention. Example 1 In this example, a device of the type shown in Figure 8 is used in which a sample flows preferentially toward the center of the immobilized receptor. A sandwich immunoassay is illustrated for the antigen human chorionic gonadotropin (hCG) in a urine sample containing a known amount of hCG. Monoclonal and polyclonal antibodies were used for the immobilized reagent and for the labelled reagent, respectively. The labelled conjugate comprises a gold sol prepared in accordance with the procedure of Faulk, W.P., et al., Immunochem. 8:1081 (1981). Briefly summarized, 100ml distilled water containing 0.01% chloro-auric acid solution is mixed with 4 ml of 1% trisodium citrate and boiled for 15 minutes. The solution turns from yellow to red at an optical density at 520nm of about 1. The bound immunological reagent is an anti-α subunit hCG monoclonal antibody supplied by Medix. A membrane 18 comprising paper-backed nitrocellulose (supplied by Schleicher and Schuell) is used. The anti-α hCG antibody is applied to the prewetted disk three times in amounts of 5µ at a concentration of 1-2mg/ml in a limited area of about 9mm diameter. After drying, the inoculated disk is then incubated with a blocking solution containing 5% BSA and 0.01% NaAzide in phosphate buffered saline, in order to decrease non-specific binding with the nitrocellulose, and placed into the device described above. Monoclonal anti-β subunit hCG antibody (supplied by Medix) is conjugated in accordance with the procedure of Roth, supra , with this gold sol and is frozen in a -50°F temperature bath. Then it is freeze-dried. A 140µl urine sample is placed in well 16, onto the upper surface of membrane 18. The sample solution is then drawn by absorbent body 24 through membrane 18, either through passages 22 or via the conduit of annular ring 23. As an example, during passage through membrane 18, the liquid flow is directed for the lower surface of membrane 18 through passageways 22 provided in septum means 20. In this way, the sensitivity of the assay is increased by causing the liquid to flow through the membrane transversely as well as vertically. In addition, the flow rate of the liquid is controlled by the size of the passages, allowing the development of an optimum incubation time for the assay reaction. This permits the visual reading of a red dot on the membrane using colloidal gold particles at antigen concentrations where this would not normally be possible. In this example, 102 clinical urine specimens (51+ and 51-) were tested and correlated 100% with the clinical findings from the specimen source. The sensitivity level was determined to be 25mIU hCG/ml. Example 2 In this instance, a device of the type shown in Figure 9 was used in which sample flows preferentially away from the center of the immobilized receptor. A sandwich assay was performed in which antigen was immobilized on the membrane, an antibody analyte of a serum sample was to be analyzed, and the labelled antibody is a conjugate of an anti-antibody to the sample antibody labelled with colloidal gold formed in the manner described above. In this instance, the anti-antibody employed was Staphylococcus aureus A protein, which serves as a universal anti-antibody. The antigen, HIV lysate (E.I. DuPont de Nemours, Wilmington, DE) at 2mg/ml in a DuPont buffer containing detergent and ethylene glycol was deposited onto a nitrocellulose membrane disk as described in Example 1. The blocking reaction was performed with 100µl of the DuPont proprietary buffer, or an equivalent buffer containing 5% BSA, 0.05% Tween-20, 0.1% NaAzide and 10% goat serum in PBS. A sample of the serum suspected of containing antibodies to the antigen is prepared by a 1:4 dilution with the DuPont buffer and 50µl is placed is well 16 and allowed to flow through membrane 18 as described above. Then membrane 18 is washed with 50µl of DuPont buffer or PBS. Thereafter 50µl of the labelled Protein A (secondary antibody), at OD₅₂₀ of 3.5, is passed through the membrane in a similar fashion, followed by an additional 50µl wash. A positive reaction is indicated by a distinct red dot on the membrane. In this example, 200 samples (31+ and 169-) were tested and correlated 100% with the reported findings from the specimen source, as determined by DuPont ELISA. The positive samples were confirmed by Western Blot analysis. Obviously, many modifications and variations of the present invention are possible after consideration of the present disclosure. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as is specifically described.

Claims: A storage and reaction apparatus for use in assays for the detection and/or determination of a bindable target substance in a liquid sample suspected of containing such substance, comprising: (a) a liquid-permeable, porous reaction membrane having an upper and lower surface, at least one defined region of said membrane or at least said upper surface thereof having immobilized thereon a receptor capable of directly or indirectly binding to the bindable target substance; (b) a body of absorbent material capable of absorbing a liquid, said body having a surface located adjacent to the lower surface of the porous reaction membrane; (c) separating means between the said lower surface of the porous reaction membrane and the said upper surface of the absorbent material body for substantially isolating liquid flow therebetween and (d) port means through said separating means for substantially directing the flow of liquid from the lower surface of said porous reaction membrane through said separating means; whereby liquid sample applied to the upper surface of said porous reaction membrane will permeate in a selected flow pattern to its lower surface and be substantially directed to at least one selected portion of the surface of said body of absorbent material. The storage and reaction apparatus of claim 1 wherein the amount of binding substance on the upper surface of said membrane is sufficient to enable the direct visual detection or determination of the target substance when bound in combination with a corresponding visible labelled substance. The storage and reaction apparatus of claim 1 or claim 2 wherein said porous membrane comprises nitrocellulose. The storage and reaction apparatus of claim 3 wherein said nitrocellulose membrane further comprises at least one liquid-permeable support material bonded in intimate contact therewith. The storage and reaction apparatus of claim 4 wherein said support material is paper, fibreglass or polyester. The storage and reaction apparatus of any one of the preceding claims wherein said port means is spaced radially from the center of the region where the receptor is immobilized and serves to direct liquid flow radially away from the center of the region where the receptor is immobilized. The storage and reaction apparatus of claim 6 wherein said port means comprises a ring or a portion thereof spaced radially from the center of the region where the receptor is immobilized. The storage and reaction apparatus of any one of claims 1 to 5 wherein said port means comprises at least one port disposed proximal to center of the region where the receptor is immobilized surrounded by said separating means and serves to direct liquid flow towards the center of the region where the receptor is immobilized. The storage and reaction apparatus of any one of the preceding claims in which said immobilized receptor is concentrated in a limited region of the upper surface of said reaction membrane. The storage and reaction apparatus of claim 9 wherein said port means is proximal to the periphery of said limited region. The storage and reaction apparatus of any one of the preceding claims wherein said separating means comprises a liquid impermeable sheet. The storage and reaction apparatus of claim 11 wherein said port means comprises at least one opening through said impermeable sheet. The storage and reaction apparatus of claim 11 wherein said port means comprises at least one port in said impermeable sheet containing liquid-permeable material. The storage and reaction apparatus of any one of the preceding claims including sealing means serving to cause substantially all of the liquid applied to the upper surface of said porous membrane to flow through the membrane in an area limited radially by said sealing means. The storage and reaction apparatus of any one of the preceding claims in which said absorbent material is pliable, said apparatus further comprising means defining said absorbent material and including a bottom wall and platform means disposed on said bottom wall projecting upwardly into said absorbent material toward the center of the region of said immobilized receptor. The storage and reaction apparatus of claim 15 wherein said platform means applies sufficient upward pressure against said absorbent material to flex said impermeable sheet and modify the unflexed shape of said port means. The storage and reaction apparatus of claim 16 in which said port means is radially spaced from the center of the region of said immobilized receptor. The storage and reaction apparatus of claim 17 wherein the interior of said absorbent material body is spread apart under the pressure created by said platform means creating channels, whereby liquid can flow through said channels outwardly from the center of the region of said immobilized receptor. An assay method for the detection and/or determination of a bindable target substance in a liquid sample suspected of containing such substances, employing as an assay device an apparatus comprising: (a) a liquid-permeable, porous reaction membrane having an upper and lower surface, at least one defined region of said membrane or at least said upper surface thereof having immobilized thereon a receptor capable of directly or indirectly binding to the bindable target substance; (b) a body of absorbent material capable of absorbing a liquid, said body having a surface located adjacent to the lower surface of the porous reaction membrane; (c) separating means between the said lower surface of the porous reaction membrane and the said upper surface of the absorbent material body for substantially isolating liquid flow therebetween and (d) port means through said separating means for substantially directing the flow of liquid from the lower surface of said porous reaction membrane through said separating means; the method comprising applying the sample, visible labelled substance, and any additional reagents to the upper surface of said permeable reaction membrane whereby the applied liquid will permeate through said port means from the membrane lower surface and be substantially directed to at least one selected portion of the adjacent surface of said absorbent material body and thereafter detecting said labelled substance bound to said upper surface as an indication of the presence or absence of target substance bound to said bindable substance on said upper surface. The method of claim 19 wherein the amount of binding substance on the upper surface of said porous reaction membrane is sufficient to enable the direct visual detection or determination of the labelled substance. The method of claim 19 or claim 20 wherein at least one of the bindable and binding substances is an immunochemical. The method of any one of claims 19 to 21 wherein said porous reaction membrane of said apparatus comprises nitrocellulose. The method of claim 22 wherein the nitrocellulose porous membrane further comprises at least one support material bonded in intimate contact therewith. The method of any one of claims 19 to 23 wherein said target substance is detected by employing a labelled substance with a label comprising colloidal gold particles or chelated gold particles.

Inventors: CHU ALBERT E, CHUN PETER K, YEUNG SIU CHIN C, CHU, ALBERT E., CHUN, PETER K., YEUNG, SIU CHIN C.
Applicants: E Y LAB INC, E-Y LABORATORIES, INC.
Application year: 1988
Main classification: G01N33
IPC codes: G01N33