(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`
`
`(43) International Publication Date
`7 April 2005 (07.04.2005)
`
`(10) International Publication Number
`
`WO 2005/031355 A1
`
`(51) International Patent Classification7:
`
`G01N 33/549
`
`(21) International Application Number:
`PCT/ US2004/03 1220
`
`(22) International Filing Date:
`22 September 2004 (22.09.2004)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/5 05 ,092
`
`22 September 2003 (22.09.2003)
`
`US
`
`(71) Applicant (for all designated States except US): QUIDEL
`CORPORATION [US/US]; 10165 MC Kellar Court, San
`Diego, CA 92121 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): LAMBOTTE, Paul
`[BE/US]; 5549 Sandburg Avenue, San Diego, CA 92122
`(US). CERRITO, Michael [US/US]; 954 Emma Drive,
`Cardiff, CA 92007 (US). RAMIREZ, Cecille [PH/US];
`26241 Palm Tree Lane, Murrieta, CA 92563 (US).
`
`(74) Agents: ZACHOW, Karen, R. et a1; Morrison & Foerster
`LLP, 3811 Valley Centre Drive, Suite 500, San Diego, CA
`92130—2332 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind ofnationalprotection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD,
`MG, MK, MN, MW, MX, MZ, NA, NI, NO, NZ, OM, PG,
`PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL, SY, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, YU, ZA, ZM,
`ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, M7,, NA, SD, SL, S7,, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, H'U, IE, IT, LU, MC, NL, PL, PT, RO, SE, SI,
`SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`with international search report
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments
`
`[Continued on next page]
`
`(54) Title: DEVICES FOR THE DETECTION OF MIILTIPIE ANALYTES IN A SAMPLE
`
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`(57) Abstract: The present invention relates generally to an assay for detecting and differentiating multiple analytes, if present, in
`a single fluid sample, including devices and methods therefore.
`
`fl]]]]]]] Control Zone(s)
` Test Zone(s)
`
`
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`For two—letter codes and other abbreviations, refer to the ”Guid—
`ance Notes on Codes and Abbreviations ” appearing at the begin—
`ning of each regular issue of the PCT Gazette.
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`WO 2005/031355
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`PCT/US2004/031220
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`DEVICES FOR THE DETECTION OF MULTIPLE ANALYTES IN A SAMPLE
`
`RELATED APPLICATION
`
`[0001]
`
`This application claims priority benefit of U.S. Provisional'Application No.
`
`60/505,092, filed September 22, 2003, which is hereby incorporated herein by reference in its entirety.
`
`TECHNICAL FIELD
`
`[0002]
`
`The present invention relates generally to an assay for detecting and differentiating
`
`multiple analytes, if present, in a single fluid sample, including devices and methods. therefor.
`
`BACKGROUND OF THE INVENTION
`
`[0003]
`
`The field of rapid diagnostic testing has evolved» for many years to permit the detection
`
`of analytes in a variety of sample types. The use of polyclonal antibodies was followed by the use of
`
`monoclonal antibodies to generate assays With high specificity for a number of analytes, including
`
`hormones, blood cells, drugs and their metabolites, as well as the antigens of infectious agents including
`
`Strep A, Strep B, Chlamydia, HIV, RSV, influenza A and influenza B and many others. The visible
`
`signal generated by enzyme—catalyzed reactions or by the accumulation of a visible signal at the level of a
`
`test line has also resulted in rapid development of highly sensitive results. Many of the rapid
`
`immunoassay-based tests include a solid housing encasing a test strip. However, recently,
`
`immunochromatographic assays have been manufactured which do not have solid housings. Such tests,
`
`referred to as dipsticks, can be dipped directly into a tube containing a pre-determined amount of the
`
`liquid sample of interest. The extremity of the dipstick containing a sample—receiving pad is generally
`
`brought in contact with a liquid sample, and the liquid migrates up the flow path. Advantages of the
`
`dipstick format include ease of use and minimum handling, which reduces the opportunities for
`
`contamination and procedural errors, and lowers manufacturing costs.
`
`[0004]
`
`One disadvantage of current immunochromato graphic dipsticks is that they can only
`
`detect the presence of a single analyte. Often these devices are limited because there is, no provision to
`
`mark the location of possible multiple test lines along the flow path. In the field of chemical urinalysis,
`
`dipsticks carrying multiple pads, each specific for a urine analyte to be detected and measured, the
`
`dipstick is dipped into the urine sample, then removed from the container, blotted to eliminate excess
`
`urine, and applied against a template in order to read the results. These devices are capable of evaluating
`
`multiple analytes, but are problematic. For example, such devices increase the chances of contamination
`
`by carry-over of material from one device to another, With the consequence of potentially inaccurate
`
`results. Moreover, this format exposes the user to potential contamination via removing the ship from
`the urine sample, blotting it on an absorbent paper, which becomes contaminated, applying it against the
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`template, which often is the exterior wall of the product container, thereby contaminating the product
`
`package itself. Further, as indicated, an external template is required to read results.
`
`[0005]
`
`It is recognized in a variety of fields, that the use of single analyte rapid tests is often
`
`limiting, for example, because only one analyte at a time can be evaluated. The advantage of rapidity is
`
`therefore challenged by the limitation of current assays as adjuncts to the diagnosis of a disease state. For
`
`instance, the pediatric units have to make differential diagnostics of Flu A, Flu B, RSV and other upper
`
`respiratory viruses, on infants that are in need of urgent care. The availability of rapid test panels would
`
`greatly facilitate the doctors’ efforts to diagnose the condition, and therefore to take the appropriate
`
`course of action faster, more easily, and at lower cost. To date, no such assay has been developed that
`
`allows the differential diagnostic of two or more analytes on a single test strip, in a minimally involving
`
`procedure.
`
`[0006]
`
`In summary, chemical urinalysis dipstick assays have been used for many years to
`
`determine the presence (or amount) of multiple analytes in a urine sample; however, the technology used
`
`to perform such assays not only has undesirable use characteristics but is not readily transferable to
`
`immunologic based assays (dipstick or lateral flow) which require flow of sample through the assay
`
`device rather than immersion of the device (in particular immersion of the test portion of the device) into
`
`a sample. For example, with respect to the use of chemical urinalysis dipsticks, the fact that they must be
`
`submerged into the urine sample, removed and blotted of excess urine then placed in physical contact (or
`
`very close proximity) with an external, typically reusable (and hence contaminable), test results panel
`
`(i.e., template) is not just undesirable but unsafe, particularly if the sample contains contagious agents
`
`such as virus or bacteria. Further, because immunologic based assays typically employ at least two,
`
`generally sequential reactions (for example a labeling followed by a capture (test) reaction), they are not
`
`amenable to submersion into a sample in the same manner as the chemical urinalysis dipsticks. Thus,
`
`there is a need in the art for devices and methods that addresses these problems in the art. The present
`
`invention addresses these and other related needs in the art.
`
`SUMMARY OF INVENTION
`
`[0007]
`
`The present invention provides analytical devices, particularly immunoassay devices,
`
`capable of determining the presence and/or amount of multiple analytesin a fluid sample, permitting
`
`more complete diagnosis or analysis of said sample. Advantageously, the present devices may be
`
`formatted as dipsticks or lateral flow devices, in either case not requiring, an external test results panel for
`
`determination of test results. By positioning the multiple test and/or control zones in predetermined
`
`patterns on the test devices in accordance with the present invention, the need for an external test results
`
`panel, or even markings on a test device housing, is eliminated. By way of example, in a preferred
`
`embodiment, a single control zone is positioned between two test zones, each test zone testing for the
`
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`presence or amount of a different analyte. It has heretofore been assumed that a control zone must be
`
`located downstream of a test zone in order function as control zone. However, the present inventors have
`
`discovered that such is not necessary and, rather, that one or more control zones may. be employed in
`
`immunoassay devices not only to provide an indication of assay completion and/or operability but also of
`
`the relative location of one or more test lines, thereby permitting rapid differentiation of analytes within a
`
`fluid sample in a single immunoassay test device.
`
`[0008]
`
`In an embodiment of the present disclosure, a device is provided for the detection of
`
`multiple analytes in a fluid sample, which device comprises: a matrix defining an axial flow path, the
`
`matrix comprising: i) a sample receiving zone at an upstream end of the flow path that receives the fluid
`
`sample, ii) a label zone positioned within the flow path and downstream from the sample receiving zone,
`
`said label zone comprising one or more labeled reagents which are capable of binding one or more
`
`analytes to form labeled analytes and are mobilizable in the presence of fluid sample, iii) one or more test
`
`zones positioned within the flow path and downstream from the label zone, wherein each of the one or
`
`more test zones contain means which permit the restraint of a different labeled analyte in each test zone
`
`or a combination of different labeled analyte in a single test zone, and wherein restrained labeled analyte
`
`is detectable within each test zone, and iv) one or more control zones positioned within the flow path and
`
`downstream from the label zone, wherein the one or more control zones incorporate means which permit
`
`the indication of the completion of an assay. In the most frequent embodiments, the device incorporates
`
`means to restrain and thereby detect two or more different labeled analytes in a sample. Also, in frequent
`
`embodiments, the device comprises a dipstick assay device. In occasional embodiments, the device
`
`comprises two or three or more test zones and two or more control zones.
`
`[0009]
`
`The present device permits the detection of multiple analytes, in a sample without
`
`reference to an external template. Moreover, frequently the device comprises a dipstick assay that lacks
`
`an external housing. In general, the analytes comprise analytes of interest and further comprise those
`
`provided herein, among others. Frequently, the present devices are useful for assaying a particular panel
`of analytes. Also frequently, the present devices are useful to simultaneously detect two or more
`
`different analytes in a sample. On occasion the present device and methods are useful to detect a panel of
`
`analytes of interest selected from an influenza panel (comprising test zones containing reagents capable
`
`of restraining a selection of influenza A, influenza B, respiratory syncytial virus (RSV), adenovirus,
`
`rhinovirus and/or parainfluenza Virus), a panel comprising one or more of streptococcus pneumoniae,
`
`mycoplasma pneumoniae and/or Chlamydia, an HIV panel, a Lupus panel, an H. Pylori panel, a
`
`toxoplasma panel, a herpes. panel, a Borrelia panel, a rubella panel, a cytomegalovirus panels, a
`
`rheumatoid arthritis panel, or an Epstein—Barr panel, among others.
`
`[0010]
`
`In one preferred aspect, each of the one or more test zones lie in fluid cormnunication
`
`with one another. Moreover, in another aspect, the one or more test zones lie in fluid communication
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`with one or more control zones. In a further aspect, the presently contemplated devices do not utilize one
`
`or a plurality of wells, rather a matrix defining an axial flow path is utilized. In a frequent embodiment, a
`
`device in accordance with the present disclosure contains a single sample receiving zone that lies in fluid
`
`communication with the one or more test zones.
`
`[0011]
`
`In frequent embodiments, the control zone is positioned between the one or more test
`
`zones. In occasional embodiments, the positioning of the control zone between the one or more test
`
`zones. comprises positioning one control zone between two test zones. Also in occasional embodiments,
`
`the positioning of the control zone between the one or more test zones comprises multiple test zones and
`
`multiple control zones, wherein each control zone is positioned between two test zones, in an alternating
`
`arrangement. Frequently, the control zone is positioned upstream of a test Zone. Also frequently, the
`
`control zone is positioned downstream of a test zone. In occasional embodiments, the control zone is
`
`positioned downstream of each or all of the one or more test zones. In another embodiment, the test and
`
`control zones are positioned in an alternating format within the flow path beginning with a test zone
`
`positioned upstream of any control zone.
`
`[0012]
`
`In one embodiment, each of the one or more test zones contain means comprising an
`
`immobilized reagent capable of specifically binding a unique analyte. Thus, in this embodiment, each of
`
`the one or more test zones contain an immobiliZed reagent capable of specifically binding a particular
`
`analyte,; wherein each of the immobilized reagents is capable of binding a different analyte than any other
`
`immobilized reagent within another test zone in the device. These means can comprise any of a variety
`
`of specific binding pair members as described elsewhere herein. On occasion, the means which permit
`
`the restraint of a different labeled analyte in each test zone comprise an immobilized capture reagent.
`
`[0013]
`
`In one aspect, the test zones can be provided in any of a variety shapes and
`
`configurations with the limitation that each particular test zone is detectably distinguishable from other
`
`test zones, if present, and the control Zone(s) in the presence of labeled analyte, if present, restrained in
`
`that test zone. In a related aspect, the control zones can be provided in any of a variety shapes and
`
`configurations with the limitation that each particular control zone is detectably distinguishable from
`
`other control zones, if present, and the test zones upon completion of an assay.
`
`[0014]
`
`In another embodiment, the label zone comprises multiple labeled reagents, wherein
`
`each of the multiple labeled reagents is capable of specifically binding a unique analyte. In occasional
`
`embodiments, the labeled reagent is a reagent capable of binding any or all of the multiple analytes, if
`
`present, in the sample. Frequently, each of the labeled reagents is detectably distinguishable from one
`
`another. Also frequently, the label component of the labeled reagent is selected from the group
`
`consisting of a chemiluminescent agent, a particulate label, a colloid label, a colorimetric agent, an
`
`energy transfer agent, an enzyme, a fluorescent agent and a radioisotope. In occasional embodiments, the
`
`labeled reagents comprise different colored labeled reagents. For example, the labeled reagent can
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`comprise 2, 3, 4, 5, or 6 or more different colored particulate reagents. Particulate label colors
`
`comprising red, blue, black, purple, and other high—contrast colors are frequently utilized in the present
`
`embodiments. In frequent embodiments, the colored particulate label comprises a colored latex
`
`particulate label. In another frequent embodiment, the colored label comprises a Carbon, Gold or
`
`Selenium colored colloid label. On occasion, the use of different colored particulate labeled reagents
`
`allows for the detection of multiple analytes via the observation of different detectable signals (e.g.,
`
`different colors) in any one or more of the one or more test zones as a result of the restraint of different
`
`labeled analyte in each test zone. In a less occasional embodiment, the labeled reagent can comprise a'
`
`mixture of any of a variety of detectable labeling schemes in one device such that each analyte can
`
`become labeled by a different labeled reagent to provide a different detectable signal.
`
`[0015]
`
`In occasional embodiments, a test zone may contain one or more immobilized reagents
`
`capable of specifically binding a unique analyte, such that multiple labeled analytes may become
`
`restrained and detectible in the test zone. In this embodiment there may be multiple analytes, of interest
`
`for the device, however, frequently only one of these analytes is present, if at all, at one time in the fluid
`
`sample. Thus, multiple labeled reagents are useful in this embodiment which are both analyte specific
`
`and detectably distinguishable (i.e., different colors). For example, a device of the present embodiment is ,
`
`capable of detecting Influenza A or influenza B antigen, if present, in a single sample. In the case of
`
`influenza A, a red-colored labeled reagent that is capable of specifically binding influenza A may be
`
`used, which would result in the development of a red—colored test zone after completion of the assay of a
`
`fluid sample containing influenza A analyte. In the case of Influenza B, a blue—colored labeled reagent
`
`that is capable of specifically binding influenza B may be used, which would result in the development of
`
`a blue-colored test zone after completion of the assay of a fluid sample containing influenza B analyte.
`
`One of skill in the art would recognize that the colors may be alternated and/or other detectibly
`
`distinguishable labeling means contemplated herein may be utilized. Frequently, such test zones can be
`
`deposited as single zones. containing a mixture of capture reagents, or as adjacent zones of single capture
`
`reagents. On occasion, in the present embodiment, multiple analytes of interest are present together in
`
`the fluid sample, which then become labeled subsequent to contact with the device and restrained in the
`
`test zone. Thus, multiple labeled analytes may be restrained in a single test zone.
`
`[0016]
`
`In another embodiment, the sample receiving zone and the label zone comprise
`
`separate components in fluid—flow contact. In a frequent embodiment, the one or more test zones and the
`
`control zone are positioned within a test region. Moreover, frequently, the sample receiving zone, label
`
`zone and the test region comprise separate components in fluid-flow contact. Also frequently, the test
`
`region comprises nitrocellulose or other material suitable for immobilization of test and control reagents,
`
`and/or is laminated on a plastic backing material. On occasion, the matrix is positioned within a housing
`
`comprising a support and optionally a cover, wherein the housing contains a sample-receiving aperture
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`and one or more observation ports. In occasional embodiments, the control zone comprises a mark that is
`
`detectable within the test region when the test region is in a moist state. In this embodiment the test
`
`region comprises a material that is opaque in a dry state and transparent in a moist state such that the
`
`mark become visible as the liquid sample moistens the test region. ‘
`
`[0017]
`
`In a further embodiment, the device is capable of detecting influenza A and/or
`
`influenza B, if present, in a single sample. In occasional embodiments, each of the multiple analytes are
`
`selected from the group consisting of a toxin, an organic compound, a protein, a peptide, a
`
`microorganism, a bacteria, 3 virus, an amino acid, a nucleic acid, a carbohydrate, a hormone, a steroid, a
`
`vitamin, a drug, an antibody, and a hapten.
`
`[0018]
`
`In another flirther embodiment, the number of control zones. is represented by the
`
`variable “11” and the number of test zones is represented by the variable “n+1,” and wherein the test zones
`
`and control zones are positioned in a series comprising an alternating format, wherein the zone arranged
`
`at the most downstream position in the series comprises a test zone. The variable “11” often refers. to 2
`
`test zones. However, on occasion, the variable “n” refers to between 2 to about 5 test zones. Thus, on
`
`occasion, 2, 3, 4, 5 or more test zones are positioned on the device. Frequently, each of the test zones
`
`permit the restraint of a different analyte. On occasion, a device is provided that is capable of detecting a
`
`number of different analytes represented by the variable “11,” wherein the number of control zones and
`
`the number of different analytes capable of being detected by the device are equal. Also on occasion, a
`
`device is provided that is capable of detecting a number of different analytes represented by the variable
`
`“n+1,” wherein the number of test zones and the number of different analytes capable of being detected
`
`by the device are equal.
`
`[0019]
`
`In frequent embodiments a device for the detection of multiple analytes in a fluid
`
`sample is provided, wherein the device comprises: a matrix defining an axial flow path, the matrix
`
`comprising: i) a sample receiving zone at an upstream end of the flow path that receives the fluid sample;
`ii) a label zone, within the flow path and downstream from the sample receiving zone, comprising a first
`
`and second labeled reagent, each of which specifically bind an analyte to form a labeled analyte and are
`
`mobilizable in the presence of fluid sample; and iii) a test region comprising a first test zone, a second
`
`test zone and a control zone, wherein the control zone is positioned between the first and second test
`
`zones Within the flow path, wherein each of the first and second test zones contain means which permit
`
`the detection of a different analyte in each test zone, and wherein the control zone incorporates means
`
`which allow for the indication of the completion of an assay. In occasional embodiments, the first zone
`
`is positioned upstream from the control zone within the flow path and the second zone is positioned
`
`downstream from the control zone within the flow path.
`
`[0020]
`
`In another embodiment, methods are provided for the detection of one or more
`
`analytes in a fluid sample. For example, a method is provided for the detection of multiple analytes in a
`
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`fluid sample, comprising: i) contacting a device of the type described above with a fluid sample
`
`suspected of containing one or more analytes, and wherein each of the one or more test zones in the
`
`device contains means which permit the restraint of a different labeled analyte or combination 'of labeled
`
`analytes in each test zone; and ii). detecting one or more labeled analytes restrained in the one or more test
`
`zones. Frequently the device comprises a dipstick-type device. In general, the analytes of interest
`
`comprise those provided herein, among others. Frequently, the present methods. are useful for assaying a
`
`particular panel of analytes. Also frequently, the present methods are useful to simultaneously detect two
`
`or more different analytes in a sample. Commonly the present devices and methods are utilized to
`
`diagnose a medical condition. Also commonly, the present devices and methods aid in guiding
`
`therapeutic decisions. On occasion, the present method steps may be practiced at different locations, and
`
`by different entities.
`
`[0021]
`
`These and other features and advantages of the present invention will be apparent
`
`from the following detailed description, examples and claims.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0022]
`
`Figure 1(A—I)‘ depicts various example configurations of the test and control zones
`
`within the presently contemplated devices. The arrow in each Figure indicates the general direction of
`
`fluid sample flow after initial contact with the device. The boxes containing vertical lines and/or the
`
`diagonal lines depict control zones, and the boxes containing crosshatched lines depict test zones. Figure
`
`1(A-J) further depicts the sample receiving Zone [1], the label Zone [2] and the test region [3.]. The
`
`present devices are not intended to be limited to the aspects indicated in the depicted embodiments, other
`
`configurations are contemplated. Moreover, the depicted aspects are not necessarily presented to scale.
`
`[0023]
`
`Figure 2 is a graph depicting the ratio of test zone and control zone signals for different
`
`volumes of sample in exemplary devices.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0024]
`
`For clarity of disclosure, and not by way of limitation, the detailed description of the
`
`invention is divided into the subsections that follow.
`
`A.
`
`Definitions
`
`[0025]
`
`Unless defined otherwise, all technical and scientific terms used herein have the same
`
`meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs.
`
`All patents, applications, published applications and other publications referred to herein are incorporated
`
`. by reference in their entirety. If a definition set forth in this section is contrary to or otherwise
`
`inconsistent with a definition set forth in the patents, applications, published applications and other
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`publications that are herein incorporated by reference, the definition set forth in this section prevails over
`
`the definition that is incorporated herein by reference.
`
`[0026]
`
`As used herein, “a” or “an” means “at least one” or “one or more.” The use of the
`
`phrase “one or more” herein does not alter this intended meaning for the terms “a” or “an.”
`
`[0027]
`
`As used herein, “disease or disorder” refers to a pathological condition in an organism
`
`resulting from,’e.g., infection or genetic defect, and characterized by identifiable symptoms.
`
`[0028]
`
`As used herein the term “sample” refers to anything Which may contain an analyte for
`
`which an analyte assay is desired. The sample may be a biological sample, such as a biological fluid or a
`
`biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool,
`sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like. Biological tissues are aggregate of
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`cells, usually of a particular kind together with their intercellular substance that form one of the structural
`
`materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium,
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`muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes,
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`arteries and individual cell(s)'.
`
`[0029]
`
`“Fluid sample” refers to a material suspected of containing the analyte(s) of interest,
`
`which material has sufficient fluidity to flow through an immunoassay device in accordance herewith.
`
`The fluid sample can be used as obtained directly from the source or following a pretreatment so as to
`modify its character. Such samples can include human, animal or man-made samples. The sample can
`
`be prepared in any convenient medium which does not interfere with the assay. Typically, the sample is
`
`an aqueous solution or biological fluid as described in more detail below.
`
`[0030]
`
`The fluid sample can be derived from any source, such as a physiological fluid,
`
`including blood, serum, plasma, saliva, sputum, ocular lens fluid, sweat, urine, milk, ascites fluid,
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`mucous, synovial fluid, peritoneal fluid, transdermal exudates, pharyngeal exudates, bronchoalveolar
`
`lavage, tracheal aspirations, cerebrospinal fluid, semen, cervical mucus, vaginal or urethral secretions,
`
`amniotic fluid, and the like. Herein, fluid homogenates of cellular tissues such as, for example, hair, skin
`
`and nail scrapings, meat extracts and skins of fruits and nuts. are also considered biological fluids.
`
`Pretreatment may involve preparing plasma from blood, diluting Viscous fluids, and, the like. Methods of
`
`treatment can involve filtration, distillation, separation, concentration, inactivation of interfering
`
`components, and the addition of reagents. Besides physiological fluids, other samples can be used such
`
`as water, food products, soil extracts, and the like for the perfomiance of industrial, environmental, or
`
`‘
`
`food production assays as well as diagnostic assays. In addition, a solid material suspected of containing
`
`the analyte can be used as the test sample once it is modified to form a liquid medium or to release the
`
`analyte. The selection and pretreatment of biological, industrial, and environmental samples prior to
`
`testing is well known in the art and need not be described further.
`
`

`

`WO 2005/031355
`
`PCT/US2004/031220
`
`[0031]
`
`As used herein, the term “specifically binds” refers to the binding specificity of a
`
`specific binding pair. “Specific pair binding member” refers to a member of a specific binding pair, i.e.,
`
`two different molecules wherein one of the molecules specifically binds with the second molecule
`
`through chemical or physical means. The two molecules are related in the sense that their binding with
`
`each other is such that they are capable of distinguishing their binding partner from other assay
`
`constituents having similar characteristics. The members of the specific binding pair are referred to as
`
`ligand and receptor (antiligand), sbp member and sbp partner, and the like. A molecule may also be a
`
`sbp member for an aggregation of molecules; for example an antibody raised against an immune complex
`
`of a second antibody and its corresponding antigen may be considered to be an sbp member for the
`
`immune complex.
`
`[0032]
`
`In addition to antigen and antibody specific binding pair members, other specific
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`bindingpairs include, as examples without limitation, biotin and avidin, carbohydrates and lectins,
`
`complementary nucleotide sequences, complementary peptide sequences, effector and receptor
`
`molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, a peptide sequence and an
`
`antibody specific for the sequence or the entire protein, polymeric acids and bases, dyes and protein
`
`binders, peptides and specific protein binders (e.g., ribonuclease, S-peptide and ribonuclease S—protein),
`
`metals and their chelators, and the like. Furthermore, specific binding pairs can include members that are
`
`analogs of the original specific binding member, for example an analyte—analog or a specific binding
`
`member made by recombinant techniques or molecular engineering.
`
`[0033]
`
`An sbp member is analogous to another sbp member if they are both capable of
`
`binding to another identical complementary sbp member. Such an sbp member may, for example, be
`
`either a ligand or a receptor that has been modified by the replacement of at least one hydrogen atom by a
`
`group to provide, for example, a labeled ligand or labeled receptor. The sbp members can be analogous
`
`to or complementary to the analyte or to an sbp member that is complementary to the analyte.
`
`[0034]
`
`If the specific binding member is an immunoreactant it can be, for example, an
`
`antibody, antigen, hapten, or complex thereof. If an antibody is used, it can be a monoclonal or
`
`polyclonal antibody, a recombinant protein or antibody, a chimeric antibody, a mixture(s) or fragment(s)
`
`thereof, as well as a mixture of an antibody and other specific binding members. The details of the
`
`preparation of such antibodies and their suitability for use as specific binding members are known to
`
`those skilled in the art.
`
`“Antigen” shall mean any compound capable