Analyte assays and devices

The present invention provides a method and a device that utilizes capillarity-mediated, chromatographic transport, for the qualitative or semi-quantitative analysis of selected analytes in liquid samples. The device utilizes an applicator/collection device for collecting and administering the sample to the flow path such that reagent(s) flow through the applicator/collection device, washing the sample into the reaction pathway. The device farther utilizes an air gap between the initial location of the reagent and the reaction pathway to funnel the reagent efficiently through the sample so as to collect all or substantially all of the sample and make it available for the reaction(s).

FIELD OF INVENTION
 The present invention is within the field of assays and devices for
 quantitative or qualitative assays of a member of any type of binding pair
 such as ligand-receptor, antigen-antibody, etc., as well as for substrates
 or products of chemical and biochemical reactions. This includes
 immunometric assays, which are designed for the analysis of selected
 analyte(s) in a sample.
 BACKGROUND OF INVENTION
 Today binding-pair assays are utilized extensively in such fields as
 clinical, forensic, and veterinary medicine, pharmacological testing,
 environmental monitoring, food quality assurance, and other related areas.
 All these fields have needs for rapid and effective analysis of specific
 substances (referred to as analytes), which are frequently found in low
 concentrations within the given test sample. The basic principles and
 mechanisms of assays are specifically designed to accommodate such urgent
 needs.
 These assays are especially useful in the in vitro determination of the
 presence and concentration of analytes within physiological fluids. For
 example, the determination of specific proteins, enzymes, hormones,
 metabolites, and therapeutic or toxic drugs within the blood, urine, or
 cerebral spinal fluid has greatly enhanced the efficacy of diagnostic
 methodologies in clinical medicine.
 Moreover, the development of non-radioactive labeling components, which
 allow the direct visualization of the completed reaction, has facilitated
 the use of binding-pair assay procedures outside of the "typical"
 laboratory. For example, in clinical office settings,
 non-radioactively-labeled, binding-pair assays are useful for providing
 rapid, simple procedures which may be performed while the patient is still
 in the office. Thus diagnosis can be accomplished without delay, and
 treatment may be instituted during a single visit.
 Without such assays, it was frequently necessary to collect the sample from
 the patient during a first visit and to have the biological sample
 analyzed by a clinical laboratory at a later time. During such time, the
 patient was sent home and often required to return for a second office
 visit in order to receive the proper treatment and/or medication. Such
 delay was at best inefficient, and at worst potentially life threatening.
 The term "binding-pair assay" refers to an assay between two binding-pair
 members designed to facilitate the formation of a complex between a
 particular analyte of interest and another substance capable of specific
 interaction with the analyte. In this way, the presence of particular
 analyte may be detected. Alternatively, the binding-pair member may be a
 substance which, if detectable, may be used to infer the presence or
 absence of the analyte within the sample.
 In the context of the present invention, the term "analyte" refers to, but
 is not limited to, compounds such as proteins, modified proteins,
 peptides, nucleic acids such as deoxyribonucleic acid (DNA), ribonucleic
 acid (RNA), peptide nucleic acid (PNA), haptens, antigens, antibodies, and
 any metabolites of these substances and any other compounds, either
 natural or synthetic, which may be of diagnostic interest and which have a
 specific binding partner (e.g., the receptor moiety of a ligand-receptor
 assay, or the substrate of an enzyme).
 Binding-pair assays rely upon the binding of one analyte by its specific
 binding partner to determine the concentration of the analyte within the
 test sample. Binding-pair assays may be differentiated and categorized as
 either competitive or non-competitive in nature. Non-competitive assays
 generally utilize the receptor component in a substantial excess over the
 concentration of the analyte to be determined in the assay.
 One type of non-competitive assay is usually referred to as "sandwich
 assay." It employs the methodology whereby the analyte is detected via its
 binding to two binding partners. One partner may be labeled to facilitate
 a subsequent detection and the other is immobilized to a solid-phase to
 facilitate separation of the bound analytes from unbound reaction
 components (e.g., unbound labeled first receptor). An alternate
 non-competitive assay can be termed a "blocking assay." In this type of
 assay, sample is first mixed with a binding partner (usually labeled), and
 any analyte in the sample binds to the binding partner. The mixture is
 then allowed to react with analyte analog, which is usually bound to a
 solid phase. The more analyte is present in the sample, the more binding
 sites on the binding partner will be blocked, and the less sites there
 will be for the analyte analog to bind. Thus, in this form of assay, the
 more label is bound to the solid phase, the less analyte is present in the
 test sample.
 In contrast, competitive binding-pair assays generally involve analyte from
 the test sample, a purified binding partner or binding partner analog that
 is labeled to facilitate detection, and a rate-limiting concentration of
 binding-partner species. The sample analyte and the labeled
 analyte/analyte analog moieties are subsequently allowed to compete for
 the limited number of binding sites provided by the binding partner
 species present in the assay mixture.
 Competitive binding-pair assays can be further differentiated as being
 homogeneous or heterogeneous in nature. In homogeneous assays, all of the
 reactants participating in the competition reaction are mixed together and
 the concentration of analyte is determined by its effect on the extent of
 binding between its binding partner and labeled analyte. The signal
 observed is a direct function of this binding and can be related to the
 overall concentration of analytes present in the test sample. U.S. Pat.
 No. 3,817,837, which is incorporated herein by reference, discloses a
 homogeneous, competitive immunometric assay in which the labeled analyte
 analog is a ligand-enzyme conjugate and the binding partner is an antibody
 capable of binding either the analyte or analyte analog. In general,
 homogeneous assay systems require both an external instrumentality to
 determine the result and the prior calibration of the observed signal by
 separate tests performed with known concentration of the specific analyte
 in a process known as standardization. While homogeneous assays have
 dominated competitive immunometric assay system development, such systems
 are not capable of providing results for the determination of multiple
 analytes in a test sample in a single-test format not requiring external
 instrumentality.
 Heterogeneous, competitive binding-pair assays require separation of the
 bound, labeled analyte or its binding partner from the free, labeled
 analyte or its binding partner and a subsequent measurement of either the
 concentration of the bound or free fraction. Methodologies for performing
 these assays are described in U.S. Pat. Nos. 3,654,090, 4,298,685, and
 4,506,009, which are incorporated herein by reference. The quantitative or
 semi-quantitative measurement of analyte concentration utilizing this
 methodology cannot be performed without the use of additional tests to
 calibrate the assay results. Hence, only the presence or absence of the
 analyte can be determined without additional instrumentation or tests.
 Recently, however, methods have been developed for the internal
 calibration of binding-pair assays by providing a device which
 incorporates reference zones whereby the given response at the reference
 zone represents the assay response for a specific concentration of the
 analyte. The response generated by the unknown concentration of the
 analyte in the test sample is then compared with the response at the
 reference zone to determine the concentration of the analyte in the test
 sample in a qualitative or quantitative manner. U.S. Pat. No. 4,540,659,
 which is incorporated herein by reference, describes the system that
 incorporates several analyte concentration standards. They provide the
 ability to make semi-quantitative determinations of analyte concentrations
 in competitive binding-pair assays through a direct visual examination.
 Sample collection and application means are known within the relevant
 field. For example, an applicator component, consisting of a separate
 wand-like component with a bibulous material attached to one end, is
 described in U.S. Pat. Nos. 5,169,789 and 4,770,853, which are
 incorporated herein by reference. The sample is collected for assay by
 simple absorption of the aqueous sample and subsequent placement of the
 collection component into the assay device. U.S. Pat. No. 4,624,929,
 incorporated herein by reference, discloses a sample collector comprised
 of a bibulous membrane confined in a housing. Collection of the sample is
 facilitated by contacting the sample collector with the desired aqueous
 sample. European Patent Application Nos. 88303744.2 and 90301697.0,
 incorporated herein by reference, disclose a wick-like sample collector,
 comprised of bibulous material, which is contiguous with the internal
 chromatography material.
 While there are numerous assay devices and sample collection and
 application means that are currently in use within the relevant fields,
 the device disclosed herein serves to mitigate several of the difficulties
 which are frequently encountered in the utilization of these devices. For
 example, the requirement for large initial sample volumes, sometimes as
 much as several milliliters, often becomes problematic with many
 commercially available devices. This volume requirement is a function of
 the comparatively inefficient sample collection and/or application means
 these devices possess. Moreover, the requirement for such large initial
 sample volumes can potentially lead to difficulties when only small sample
 volumes or only a single test sample is available.
 In contrast, due to the utilization of a novel sample collector and
 applicator, the present invention requires comparatively small initial
 sample volumes for analysis of analyte concentration. The features of the
 present invention also negate the need for secondary, external sample
 collector/applicator, which many, if not all, of the currently utilized
 devices require. Furthermore, the present invention greatly reduces the
 probability of sample contamination or cross-contamination, which is
 frequently encountered with the use of such secondary means. An additional
 unique feature of the sample collector/applicator component of the present
 device is the ability to collect and solubilize a dried sample, which
 putatively contains the analyte, without the use of secondary
 instrumentalities or methodologies.
 SUMMARY OF THE INVENTION
 The present invention is directed to any type of binding-pair assay and
 device that utilizes chromatographic, capillarity-mediated transport for
 the qualitative or quantitative analysis of selected analytes in samples.
 The disclosed assay system is comprised of a chromatography device which
 incorporates a sample collection and application component for the
 delivery of the solubilized sample directly into the chromatographic flow.
 The disclosed device fulfills numerous, unmet needs within the relevant
 fields. It provides, but is not limited to, the following, benefits:
 (1) All required components of the assay system are contained within a
 single unitized device that is capable of being disassembled into a sample
 collector/applicator and detection means;
 (2) the assay system allows the collection of the sample and its subsequent
 application to the chromatographic components in such a way as to minimize
 any potential sample diminution due to incomplete sample delivery;
 (3) the assay system allows collection of a comparatively small sample
 volume;
 (4) the assay system negates the requirement of an additional external
 instrumentality which collects and applies the sample to the assay device;
 (5) the assay system minimizes the potential for sample contamination
 and/or cross-contamination caused by the use of non-integrated, external
 sample collection and/or application devices; and
 (6) one embodiment of the present assay system discloses the use of
 self-contained, breakable, reagent containers for the delivery of various
 solutions utilized in the analysis of the sample.
 Currently, a major problem associated with binding-pair assays is the
 collection of the test sample and its subsequent application to the assay
 device. Many, if not all, of the commercially available devices require
 comparatively large sample volumes to be collected because the devices are
 inefficient in delivering the applied sample to the chromatographic means.
 The present device, however, greatly mitigates these difficulties via a
 novel sample applicator, which utilizes an absorbent wick-like component
 and air gap to facilitate sample delivery to the associated
 chromatographic components contained within the device. Therefore, the
 sample putatively containing the analyte of interest can be transferred to
 the chromatographic means by having the reagent(s) flow through the sample
 collector/applicator, concomitantly washing the sample onto the
 chromatographic means.

DETAILED DESCRIPTION OF THE INVENTION
 The present invention provides binding-pair assays and devices that utilize
 capillarity-mediated, chromatographic transport, for the qualitative or
 quantitative analysis of selected analytes in samples. The invention is
 useful for a wide variety of assays, both ligand-based and
 non-ligand-based. Applicable ligand-based methodologies may include, but
 are not limited to, competitive immunoassays, non-competitive or so-called
 sandwich technique immunoassays, and blocking assays.
 The use of the invention is not limited to immune systems. Other ligand
 assays include ligand-receptor assays, such as detecting insulin with an
 insulin receptor, and ligand-binding protein type assays, such as
 detecting certain antibiotics using a beta-lactam binding protein. Enzymes
 and their substrates can be determined as well. Applicable
 non-ligand-based assay methodologies may include any type of chemical and
 biochemical reactions that can be detected by a device that utilizes
 chromatographic, capillarity-mediated transport.
 The present device comprises a housing which includes means for the
 collection of a sample putatively containing the analyte of interest, its
 application into the assay device and means for the detection, such as by
 direct visualization of the results of the binding-pair assay. The device
 may also further comprise
 (1) a housing with means enclosed therein for the conjugation of a label to
 a specific analyte to be detected within the sample (label transfer pad);
 (2) a means for the sequestering the specific analyte within a defined
 spatial location (capture zone); and
 (3) a means for allowing liquids and any non-sequestered, solubilized
 materials contained therein, to undergo capillarity-mediated transport
 away from the capture location for subsequent absorption in a discrete
 area (blotter pad).
 The present invention has broad application. It may also be employed in any
 number of assays which utilize bibulous material to mediate the flow away
 from an initial spatial location where the bibulous material is contacted
 with a medium putatively containing either the analyte to be determined or
 reagents used for the analysis of the analyte.
 Prior to proceeding with the detailed description of the specific
 embodiments of the present invention, a number of terms will be defined.
 Definitions
 Analyte: the substance or composition to be measured in the assay.
 Analyte analog: a specific derivative of the target analyte which may be
 optionally attached, either covalently or non-covalently, to other
 chemical species (e.g., a label). The analyte analog may be used, for
 example, to compete with the analogous target analyte for binding to the
 specific binding partner (i.e., competition assay). Where the modification
 of the analyte provides means to join the analyte analog to another
 molecule, or where the analyte has a functionality which is used to bind
 directly to another molecule, the analyte portion of the conjugate will be
 referred to as an analyte analog.
 Binding partner: a molecule, such as a receptor, binding protein, antibody
 or antibody fragment, or enzyme (which binds to its substrate), which
 possesses the ability to interact with another molecule in a highly
 specific polar and spatial manner.
 Antibody: an immunoglobulin, or a derivative or fragment thereof, which is
 capable of specifically binding to an antigen in a receptor-ligand based
 reaction. The antibody or fragment may be polyclonal or monoclonal, or
 native or chimeric. The preparation of antibodies is well known in the
 art. For example, antibodies may be generated by the immunization of a
 host followed by the collection of sera (polyclonal), or by hybridoma cell
 line-based technology (monoclonal).
 Label: any molecule which is bound (via covalent or non-covalent means,
 alone or encapsulated) to another molecule or solid support and which is
 chosen for specific characteristics which allow detection of the labeled
 molecule. Generally, labels are comprised of, but are not limited to, the
 following types: particulate metal and metal-derivatives, radioisotopes,
 catalyticor enzyme-based reactants, chromogenic substrates and
 chromophores, fluorescent and chemiluminescent molecules, and phosphors.
 The utilization of a label produces a signal which may be detected by
 means such as detection of electromagnetic radiation or direct
 visualization, and which can optionally be measured.
 Bibulous material: a porous material that is susceptible to traversal by a
 liquid medium in response to capillary force. Such materials can be
 hydrophilic, or are capable of being rendered hydrophilic, and include
 natural polymeric substances (e.g., cellulosic materials),
 fiber-containing papers (e.g., filter and chromatographic papers), and
 synthetic or modified naturally-occurring polymers (e.g., nitrocellulose,
 cellulose acetate, polyacrylamide, cross-linked dextrose., or agarose),
 which are either utilized by themselves or in combination with other
 materials. The bibulous material may be poly-functional, or be capable for
 being made poly-functional, for example to permit covalent bonding of
 receptors, antibodies, or other compounds which function as components of
 the specific assay methodology.
 Capillary Communicating Contact: When two elements of the device are in
 capillary communicating contact, the elements of the device are capable of
 transferring fluid from one element to the other, when fluid is present,
 by capillary action.
 The Device
 The present invention provides a methodology and device for any types of
 binding-pair assays, utilizing capillarity-mediated, chromatographic
 transport for the qualitative or quantitative analysis of a selected
 analyte putatively contained within a test sample.
 FIGS. 1-6 depict a device for the detection of a specific analyte in a
 sample comprising two distinct components: the Sampler (1) and the
 Detection (2) members. The Sampler member (1) is comprised of an absorbent
 wick or tubing (6) which exhibits capillarity, and a reagent delivery
 system (22). The wick or tubing (6) is enclosed in a housing (4)
 fabricated from a rigid or semi-rigid, non-water-permeable material. When
 a wick is used for element 6, it is comprised of a bibulous material. When
 tubing is used for element 6, it may be capillary tubing of glass,
 plastic, or other appropriate material.
 The reagent delivery system (22) can be contained within the housing (4),
 partially within and partially outside the housing (4), or fully outside
 the housing (4). Optionally, an absorbent reagent pad (3) is enclosed
 within the housing (4) as a part of the reagent delivery system (22). An
 opening, the reagent application port (5), can be optionally provided in
 the housing (4) of the Sampler member to facilitate the addition of
 various solutions through the reagent delivery system (22), optionally
 through the absorbent reagent pad (3).
 The wick or tubing (6) is in capillary communicating contact with the
 reagent delivery system (22) via the absorbent reagent pad (3) when it is
 present, or via any other source of reagent. A line of demarcation (7) can
 be utilized on the absorbent wick or tubing (6) to facilitate the
 collection of a pre-determined volume of a liquid sample via capillary
 action.
 The Detection member (2) is comprised of a housing, fabricated from a rigid
 or semi-rigid, non-liquid-permeable material, optionally providing an
 upper (8) and lower (9) section. The housing can serve to both contain and
 position various components of the assay device.
 Contained within the housing is the chromatography region (24), utilizing a
 chromatography medium (10) which is comprised of at least a transit zone
 (20) and a capture zone (13). The transit zone (20) can optionally include
 a proximally located label transfer pad (11). When utilized, the label
 transfer pad (11) contains a labeled, specific, analyte-binding reagent
 (L-SABR) or the components and means for assembling an L-SABR (for a
 noncompetitive reaction) or a labeled, specific, analyte analog (L-SAA) or
 the components and means for assembling an L-SAA (for a competitive
 reaction). If components and means for assembling the L-SABR or L-SAA are
 used, the components must be capable of assembling and interacting in the
 form of an L-SABR with the analyte, or in the form of an L-SAA with the
 binding partner. The L-SABR or L-SABR, or its components, is retained
 within the label transfer pad (11) while the absorbent material comprising
 the pad is in the dry state. When components and means for assembling an
 L-SABR or L-SAA are utilized, L-SABR or L-SAA is assembled prior to or
 when analyte moves through the transfer pad (11) during the course of the
 assay. The L-SABR or L-SAA (either initially present or subsequently
 assembled) becomes freely mobile through both the label transfer pad (11)
 (when used) and the porous chromatography medium (10) when contacted by
 the assay liquid.
 Alternatively or additionally, label and/or components and means for
 assembling the L-SABR or L-SAA can be added through a detection port (19)
 which enables detection in the Detection member (2) optionally located at
 the proximal end of the transit zone (20). The L-SABR or L-SAA thus added
 or assembled enters the transit zone and becomes freely mobile through
 both the label transfer pad (11) (when used) and the porous chromatography
 medium (10) when contacted by a solution.
 The detection port (19) can also be used for the addition of buffer(s),
 reagent(s) and/or other reaction components to be used in the assay. For
 example, a reagent useful in visualizing the L-SABR or L-SAA can be added
 through the detection port (19); likewise, a buffer for solubilizing
 L-SABR or L-SAA entrapped in the label transfer pad (11) can be added
 through the detection port (19).
 The chromatography medium (10) can optionally be in capillary communicating
 contact with a distally located blotter pad (12). The chromatography
 medium (10) comprises a spatially-distinct location defined as the capture
 zone (13), said zone being located downstream from the transit zone (20)
 and the optional label transfer pad (11) and/or the optional detection
 port (19) and upstream from the blotter pad (12). A non-reagent-soluble,
 non-labeled specific analyte-binding reagent (SABR), or a specific analyte
 analog (SAA) is immobilized on the label transfer pad (11) when it is
 used. The L-SABR and the SABR can have the same or different reactive
 sites, depending on the type of assay to be performed. Likewise, the L-SAA
 and the SAA can have the same or different reactive sites.
 In one possible embodiment, the assay of the sample putatively containing
 the analyte of interest is initiated by a separation of the Sampler (1)
 and Detection (2) members from one another. The distal end portion (16) of
 the absorbent wick or tubing (6) is next brought into contact with the
 chosen liquid sample (e.g., a blood droplet). The sample is drawn into the
 absorbent wick or tubing (6), via capillary action, until such time as the
 desired amount of sample has been collected (for example, until the level
 of the collected liquid sample has reached the line of demarcation (7)).
 Optionally, a mechanism capable of puncturing skin to draw blood or other
 fluids (a "sharp") (23) can be incorporated in the device. Preferably this
 sharp is retractable or can be easily removed from the sampler member (1)
 after puncture. FIG. 6 shows two versions of a retractable sharp. In
 another embodiment, illustrated in FIG. 7, the sharp can itself be concave
 or hollow and can surround the wick or tubing (6). The sample can then
 flow along the sharp body and be exposed to the distal end of the wick or
 tubing (6) to allow collection of the sample by the wick or tubing (6).
 Alternatively, the concave or hollow sharp can be positioned next to the
 wick or tubing (6) such that the sample will flow along the sharp body and
 then encounter the proximal end of the wick or tubing (6) at the base of
 the sharp, such as through a hole in the sharp. The sample would then
 proceed to flow through the wick or tubing (6).
 The assay device is then reassembled by the insertion of the Sampler member
 (1) into the Detection member (2), such that the absorbent wick or tubing
 (6) is placed in capillary communicating contact with the chromatography
 medium (10) present at the proximal end of the label transfer pad (11). A
 solution (e.g., buffer) is then added to the reagent application port (5)
 such that the absorbent reagent pad (3) becomes saturated. The solution is
 drawn into the absorbent wick or tubing (6) by its inherent capillarity,
 causing the movement of the test sample through the absorbent wick or
 tubing (6) and into the label transfer pad (11).
 An air gap (14) is utilized to separate the absorbent reagent pad (3) from
 the label transfer pad (11), with the air gap (14) being "bridged" by the
 absorbent wick or tubing (6) upon reassembly of the assay device. The
 utilization of the air gap (14) maximizes the transfer of the liquid
 sample contained within the absorbent wick or tubing (6) to the label
 transfer pad (11). It forces capillarity-mediated transfer of the liquid
 from the absorbent reagent pad (3) through the absorbent wick or tubing
 (6), concomitantly facilitating sample transfer.
 In this embodiment, the movement of the test sample and liquid reagent into
 the label transfer pad (11) causes the L-SABR or L-SAA to be solubilized,
 thus facilitating the formation of a complex between this labeled moiety
 and any specific analyte putatively contained within the test sample. When
 L-SABR is used, the analyte/L-SABR complex subsequently interacts with,
 and is sequestered by, the immobilized, non-labeled SABR located within
 the capture zone (13). Depending upon the type of label chosen, detection
 of this sequestered complex may be accomplished by direct visualization
 via the transparent viewing port (15) located within the upper section (8)
 of the Detection member (2) over capture zone (13) to allow visualization
 of the capture zone (13).
 Any dissolved, non-complex and non-sequestered materials found within the
 capture zone can be minimized by a continuous, capillary-based flow of the
 solution (e.g., buffer) applied to the absorbent reagent pad (3) which
 washes these non-sequestered components past the capture zone (13).
 Generally, this flow will continue until either the absorbent reagent pad
 is depleted or the blotter pad (12) becomes saturated.
 In another embodiment, illustrated in FIG. 4, the device incorporates one
 or more self-contained, breakable reagent container(s) (17). The reagent
 container (17) encloses a solution which, when released, flows into and/or
 through the absorbent reagent pad (3). This solution may optionally
 contain a variety of substances including, but not limited to, analyte,
 analyte analog, specific analyte -binding reagent, signal generating
 reagent (e.g., a substrate for an enzymatic label), or any other ancillary
 reagent. The container may be integral with the housing of the device,
 separate therefrom, or both, where more than a single self-contained
 reagent container is employed.
 The device may optionally comprise a reagent application port (5), so that
 at least one of the breakable containers may be positioned so as to allow
 fluid flow through the reagent application port (5) upon rupture of the
 container(s) (17). Upon rupture, the enclosed solution is rendered capable
 of undergoing capillarity-mediated transport through the optional
 absorbent reagent pad (3) and the absorbent wick or tubing (6).
 The reagent containers (17) themselves are preferably water-impermeable
 before breakage, and may be rigid, semi-rigid, or flexible. The solution
 contained therein is capable of being delivered to the assay device by
 crushing, cutting, puncturing, melting, or otherwise rupturing the
 container (17) or a seal between the container and the assay device.
 Materials utilized in the fabrication of the reagent containers may
 include, but are not limited to, glass, polymers, fiber-reinforced papers,
 plastics, waxes, and other materials which meet the previously discussed
 structural criteria. The container shape may be any shape which is
 spatially and sterically compatible with the present device (e.g.,
 spherical, rectangular, ellipsoidal, and so-forth). The total volume of
 the reagent container (17) will vary, depending upon the specific liquid
 reagent contained therein, the function of the given reagent in the assay,
 the overall size of the assay device, the total absorptive capacity of the
 bibulous materials utilized in the assay device, the total number of
 reagent containers employed, and similar limitations.
 The housing comprising either or both the Sampler (1) and Detection (2)
 components of the assay device may be fabricated from various
 moisture-impermeable materials including, but not limited to, thermo- and
 vacuum-formed plastics, fiber-reinforced paper products, polymers and
 other appropriate materials. The material utilized to fabricate the
 housing preferably does not interfere with the sample, the sample medium,
 or any reagents used in the assay procedure. While a transparent material
 can be used in the fabrication of the viewing port (15), alternative
 embodiments include no covering on the viewing port (15), or a large
 portion of the upper section (8) of the Detection component (2) being
 fabricated from transparent material.
 Following the insertion of the various chromatographic materials during the
 construction of the device, the upper (8) and lower (9) sections of the
 Detection component (2) may be securely fastened together by ultrasonic
 welding, adhesives, and other relevant methodologies. Alternately, the
 Sampler (1) and/or the Detection (2) components may each be fabricated in
 a single, unitized piece, which is subsequently used to enclose the
 various internal components.
 Both the absorbent reagent pad (3) and blotter pad (12) may be constructed
 from any bibulous, porous, or fibrous material that is capable of
 absorbing a liquid. Examples include porous plastic polymer materials
 including, but not limited to, polypropylene, high molecular weight
 polyethylene, polyvinylidene fluoride, acrylonitrile, and
 polyterafluoroethylene; cellulosic materials (e.g., nitrocellulose); or
 heavy-weight, high-absorbency chromatographic paper. The use of a
 chromatographic paper in the fabrication of the blotter pad (12) is
 generally preferable, due to its high degree of absorbency and low cost.
 Additionally, with respect to the absorbent reagent pad (3), it is
 preferred that the chosen material retains some degree of structural
 integrity when saturated. Regardless of the material chosen, it may be
 advantageous to pre-treat the member with a surface-active agent during
 fabrication so as to reduce any inherent hydrophobicity and concomitantly
 increase its ability to absorb and deliver liquid samples in an
 efficacious manner.
 It should also be noted that the proximal end (16) of the absorbent wick or
 tubing (6) member may be saturated with an inert dye (e.g., Blue Dextran)
 to facilitate the visualization of the chromatographic flow of colorless
 solutions.
 Chromatographic media (10) which may be utilized with the present device
 include those chromatographic substrate materials possessing capillarity
 and the capacity for chromatographic solvent transport of non-immobilized,
 liquid-soluble reagents and sample components. while a wide variety of
 chromatographic materials used for paper chromatography are suitable for
 use with this invention, the use of microporous or microgranular
 thin-layer chromatographic substrates is generally preferred due to the
 marked increase in speed and resolution which these materials provide.
 The chromatographic material is preferably inert, as well as physically and
 chemically non-reactive with any of the sample components, reagents, or
 reaction products. Preferably, microporous nitrocellulose material may be
 utilized with a high degree of efficacy in the disclosed device.
 Nitrocellulose has the added advantage that the specific analyte-binding
 reagent (e.g., antibody) present in the capture zone may be immobilized
 without prior chemical treatment.
 In contrast, if the chromatography material is comprised of a
 chromatographic paper, for example, the immobilization of the specific
 analyte-binding reagent can be performed via chemical coupling
 methodologies. Commonly used reagents include, but are not limited to,
 cyanogen bromide (CnBr), carbonyldiimididazole, or tresyl chloride.
 Additionally, reagents capable of blocking non-specific binding sites on
 the chromatographic medium which might hinder chromatographic solvent
 transport of the liquid sample or reagents may also be employed in the
 fabrication of the present device. Commonly used reagents include, but are
 not limited to, bovine serum albumin (BSA), gelatin, and casein, which are
 preferably selected for their ability to not interfere with, or
 cross-react with, the sample components, reagents, or reaction products.
 SABR which may be utilized with the present invention are readily
 identifiable to one of skill in the relevant art and include those
 materials which are members of a "specific binding pair" (e.g., ligand and
 receptor, antigen and antibody, or enzyme and substrate). For example, an
 analyte and a receptor are related in that the receptor specifically binds
 to the analyte and possesses the capacity to differentiate the analyte
 from other materials having similar characteristics. The methodologies and
 devices disclosed by the present invention are particularly useful in the
 practice of immunological assay techniques wherein the specific
 analyte-binding reagents consist of receptors, antibodies, antibody
 fragments, or synthetic antibodies or antigens. The present device
 preferably employs a specific binding pair consisting of the desired
 analyte and an antibody which is specific for, i.e., has a high affinity
 for binding to, said analyte.
 The "label" utilized on the L-SABR or L-SAA disclosed in the present
 invention may be selected from, but is not limited to, the following
 categories: chromogens/fluorescers, particulate metals or their
 derivatives, components of catalyzed or enzymatic reactions,
 chemiluminescent compounds, and radioactive isotopic labels.
 Chromogens include those compounds which absorb light in a distinctive
 range such that a specific color may be visibly observed (i.e., dyes), or
 emit light when irradiated with electromagnetic radiation of a specific
 wavelength or wavelength range (i.e., fluorescent compounds). Illustrative
 dye types include, but are not limited to, quinoline, acridine, alizarin,
 cyanine and anthraquinoid dyes. Fluorescent compound functional groups
 include, but are not limited to, porphyrins, 2-aminoaphthalene,
 p,p'-diaminobenzophenone imines, 1,2-benzophenazin, and quaternary
 phenanthridine salts. By irradiating a fluorescer with light of a specific
 wavelength, one may obtain a plurality of emissions, thus providing
 multiple measurable events.
 Particulate metal sol-based label can be obtained by the direct or indirect
 coupling of the desired reaction component (e.g., antibody) with particles
 of an aqueous dispersion of a metal, metal compound, or polymer nuclei
 coated with a metal or metal compound, having a particle size of at-least
 5 nm. The term "coupling" is understood within the relevant art to
 encompass any type of chemical or physical binding, and includes covalent
 and hydrogen bonds, polar attraction, absorption, and adsorption. Metals
 utilized in this labeling methodology may include, but are not limited to
 gold, platinum, silver, copper, and iron. A metal sol is generally defined
 as a suspension of metal or metal-derivative particles which, due to their
 extremely small diameter, remain in a suspension solely because of the
 Brownian motion effects. The particulate metal sols to be used as labels
 may be prepared in a number of ways which are in themselves known. For
 example, the preparation of a gold sol has been described (G. Frens, 241
 Natural Physical Science 20 (1973)), which is incorporated herein by
 reference.
 Catalyzed reactions may be either enzyme or non-enzyme based.
 Considerations which must be taken into account when using an enzyme-based
 label include, but are not limited to, enzyme stability, turnover rate,
 sensitivity of the reaction to environmental factors, the nature of the
 substrate and products, and the effect of conjugation on the enzyme's
 catalytic properties. The methodologies involved in enzymatic labeling are
 well known within the relevant art. A preferred example is horseradish
 peroxidase (HRPO), used in conjunction with 1,7-dihydroxynaphthalene, and
 4-amino-antipyrine hydrochloride.
 An alternative form of labeling is the use of chemiluminescent compounds.
 The chemiluminescent source comprises a compound that becomes
 electronically excited following a chemical reaction and subsequently may
 either emit light in the visible range or donate energy to a secondary
 fluorescent acceptor compound. These include, but are not limited to, the
 2,3-dihydro-1,4-phthaiazinedione family (luminol), the
 2,4,5-triphenylimidazole family (lophine), and the para-dimethylamino
 oxalate-ester family (luciferase).
 Radioisotopic labels are widely utilized in immunometric assays. The
 radiolabel may be selected from, but is not limited to, the following
 radioactive isotopes: .sup.3 H, .sup.14 C, .sup.32 P, .sup.125 I and
 .sup.131 I. Methods of labeling substances with radioactive labels are
 well known within the relevant art.
 In the present invention, the utilization of metal sol-based labels is
 preferred. For example, a gold sol label conjugated to the specific
 analyte-binding reagent (e.g., antibody) may be utilized. This label
 allows the end-point of the reaction to be visualized without the need for
 any additional instrumentality.
 The present invention clearly fulfills several unfulfilled needs within the
 field of binding-pair assays. The novel construction of the unitized
 Sampler member facilitates both the initial collection, and subsequent
 assay of the sample putatively containing the analyte. The utilization of
 a novel absorbent wick/air gap feature in the Sampler component not only
 minimizes the potential for the contamination and cross-contamination of
 the sample by not requiring a secondary external means for sample
 collection (e.g., via syringe, pipette, capillary tube, etc.), but also
 maximizes the transfer of the sample to the assay device, thus minimizing
 the required sample volume.
 The following specific embodiments, by-way of example, are descriptions of
 the utilization of the disclosed invention for the purpose of immunometric
 determination of a specific analyte present in a sample. These examples
 are not meant to limit the scope of the present invention in any manner,
 but instead, merely serve to illustrate some of many possible embodiments.
 Example 1
 With reference to FIGS. 1-3, the assay is begun by a separation of the
 Sampler (1) and Detection (2) components from one another. The distal end
 (16) of the absorbent wick (6) is next brought into contact with the
 sample, a blood droplet. The sample is then drawn into the absorbent wick
 (6) via capillarity-mediated transfer until such time as the fluid level
 has reached the line of demarcation (7), thus providing a means for the
 collection of a pre-determined sample volume. The assay device is then
 reassembled by the insertion of the Sampler component (1) into the
 Detection component (2), such that the absorbent wick (6) is placed in
 capillary communicating contact with the label transfer pad (11). A
 solution is then added to the reagent application port (5) such that the
 absorbent reagent pad (3) is saturated. The solution is subsequently drawn
 into the absorbent wick (6) by its inherent capillarity, thus causing the
 movement of the sample through the absorbent wick (6) and into the label
 transfer pad (11). The label transfer pad (11) contains a particulate
 metal label which is conjugated to an antibody, with the antibody being
 specific for the analyte putatively contained within the test sample. The
 particulate metal label/antibody conjugate ("labeled antibody") is found
 in a dried, non-immobilized form within the label transfer pad (11). An
 air gap (14) is utilized to separate the absorbent reagent pad (3) from
 the label transfer pad (11), and is bridged by the absorbent wick (6) upon
 reassembly of the assay device. The concomitant movement of the test
 sample and solution into the label transfer pad (11) causes the labeled
 antibody to be solubilized, facilitating the formation of a complex
 between the labeled antibody and any specific analyte putatively contained
 within the test sample. The analyte/labeled antibody complex subsequently
 interacts with, and is sequestered by, the immobilized, non-labeled
 antibody located within the capture zone (13). Detection of this
 sequestered complex is visualized via a transparent viewing port (15)
 located within the upper section (8) of the Detection component (2) and
 over the capture zone (13). Any liquid soluble, non-reacting materials
 found within the capture zone (13) are minimized by a continuous,
 capillary-based flow of the solution initially applied to the absorbent
 reagent pad (3). This flow continues until either the absorbent reagent
 pad (3) is depleted of solution or the blotter pad (12) becomes saturated.
 Example 2
 With reference to FIGS. 1-3, the device is utilized as in Example 1 with
 the exception that the labeled antibody has been localized to the distal
 end-portion (16) of the absorbent wick (6) rather than to the label
 transfer pad (11). This specific embodiment allows for the pre-incubation
 of the sample with the labeled antibody prior to both the reassembly of
 the Sample (1) and Detection (2) components and the subsequent addition of
 a solution (e.g., buffer) to the reagent application port (5). This
 specific embodiment ensures that the labeled antibody reaches the test
 sample, by the process of diffusion, in gradually decreasing
 concentrations, due to the solubilizing of the labeled antibody following
 the addition of a solution (e.g., buffer) to the reagent application port
 (5). The timing between the initial collection of the test sample and the
 subsequent reassembly/solution addition may be varied to allow for the
 adjustment of assay sensitivity.
 Example 3
 With reference to FIGS. 1-3, the device is utilized as in Example 1 with
 the exception that the labeled antibody has been localized to the proximal
 end-portion (18) of the absorbent wick (6) rather than to the label
 transfer pad (11). This specific embodiment ensures that the labeled
 antibody reaches the test sample, by the process of diffusion, in
 gradually increasing concentrations, as opposed to the previous example
 (Example 2) where the labeled antibody reaches the test sample in
 gradually decreasing concentrations, due to the solubilizing of the
 labeled antibody following the addition of a solution (e.g., buffer) to
 the reagent application port (5). Requirements for differing reaction
 kinetics may favor one embodiment over the other.
 Example 4
 With reference to FIGS. 1-3, the device is utilized as in Example 1 with
 the additional step of localizing a chromogenic compound (e.g.,
 liquid-soluble dye) to the distal end-portion (16) of the absorbent wick
 (6). This specific embodiment allows for the direct visualization of
 capillarity-mediated transfer of the test sample through the
 chromatography medium (10), thus facilitating monitoring of
 difficult-to-detect samples (e.g., colorless aqueous samples or various
 physiological fluids).
 Example 5
 With reference to FIGS. 1-3, the device is utilized as in Example 1 with
 the exception that following the initial disassembly of the assay device
 by the removal of the Sampler component (1) from the Detection component
 (2), and prior to the actual collection of the test sample, a solution
 (e.g., buffer) is added to the reagent application port (5). This causes
 the saturation of the absorbent wick (6) member and allows for the
 solubilizing and capillarity-mediated, transfer of a dried or semi-dried
 sample onto the distal end-portion (16) of the absorbent wick (6).
 Example 6
 With reference to FIGS. 1-3, the device is utilized as in Example 1 with
 the exception that after the actual collection of the test sample, a
 solution with an added reagent such as analyte or analyte analog is added
 to the reagent application port (5). This causes the saturation of the
 absorbent wick (6) member and allows for the solubilizing and
 capillarity-mediated transfer of a dried or semi-dried sample onto the
 distal end-portion (16) of the absorbent wick (6).
 Example 7
 With reference to FIGS. 1-4, the device is utilized in a manner so as to
 provide a competitive immunoassays. The competitive assay may be
 homogeneous or heterogeneous in nature. In a homogeneous assay, all of the
 reactants participating in the competition reaction are mixed together and
 the concentration of the analyte is determined by its effect on the extent
 of binding between the receptor and labeled analyte analog. The observed
 signal is a direct function of this binding and can be related to the
 overall concentration of analyte present in the test sample.
 In a heterogeneous competitive binding-pair assay the bound, labeled
 analyte or receptor is separate from the free, labeled analyte or
 receptor. The concentration of the bound or free fraction is subsequently
 measured.
 In this example, the analyte analog may be added to the device by direct
 application to the reagent application port (5) using an external delivery
 device, or, alternately, it may be contained within the reagent containers
 (17) which, when ruptured, deliver the analyte analog to the device. The
 analyte analog may be delivered to the device either before or after the
 actual collection of the test sample. Alternatively, it may be contained
 in the wick or tubing (6).
 Example 8
 In addition to ligand-based assays, this type of device is also useful for
 non-ligand assay methodologies such as to quantify blood glucose in
 diabetes. With reference to FIGS. 1-3, the device is utilized as in
 Example 4. Test sample potentially containing .beta.-D glucose is assayed,
 and glucose oxidase is the solution added to the reagent application port
 (5). Glucose oxidase reacts with .beta.-D glucose in the presence of
 oxygen to produce D-glucose acid and H.sub.2 O.sub.2. Subsequently,
 horseradish peroxidase (HRPO), 1,7-dihydroxynaphthalene, and
 4-amimoantipyrine hydrochloride are added to the distal end-portion (16)
 of the absorbent wick (6) as chromogenic compounds. Alternatively, the
 compounds can be added before or during the assay through the detection
 port (19) to have the HRPO system present on the label transfer pad (11),
 or can be located directly on (a) the label transfer pad (11), (b) the
 chromatography medium (10), or (c) the capture zone (13).
 Example 9
 This device can also be used to puncture skin to draw blood or other fluids
 for the assay to be run in the device. With reference to FIGS. 6A and 6B,
 the retractable sharp (23) is extended, used to perforate the skin, and is
 then either retracted or broken off and disposed of. The sample is then
 collected and assayed as described in the previous examples.
 Example 10
 In another embodiment of the device, as illustrated in FIG. 7, the sharp
 (23), which is concave or a hollow tube, is used to perforate the skin.
 The sample then flows through the sharp to the wick or tubing (6). The
 sample is then assayed as described in the previous examples.
 While embodiments and applications of the present invention have been
 described in some detail by way of illustration and example for purposes
 of clarity and understanding, it would be apparent to those individuals
 skilled within the relevant art that many additional modifications would
 be possible without departing from the inventive concepts contained
 herein.