US20070087451A1 - Immuno-gold lateral flow assay - Google Patents

Abstract

A chromatographic lateral-flow assay system for rapid, high sensitivity method of detecting low levels of ligands in body fluids, with few false positives and few false negatives. The lateral-flow assay may have a membrane strip in ribbon form, which increases detection on the order of 2 to 10 fold over the conventional chromatographic specific binding assay techniques by placing a dried or lyophilized conjugate in colloidal spheres opposite side of the lateral flow membrane strip. A chromatographic specific binding assay strip device, comprising: a laminate strip having a first side and an second side; a conjugate pad or membrane disposed on said first side of said laminate; a hinge region connecting sample receiving pad or membrane strip and reservoir pad or membrane disposed on said second side of said laminate; wherein said laminating material isolates the conjugate pad from the sample pad such as to slow the fluid path into the fibrous sample pad, and a detection pad or membrane strip disposed between the sample pad or membrane and the reservoir pad or membrane on said first side of said laminate. The more complete mixing permitted by this temporary obstruction provides an important feature of the invention that gives this 2-step test its superior performance. The assay system comprises a housing device, such as a test tube or cassettes to facilitate the mixing of a sample solution with the dried or lyophilized conjugate, and kits.

Description

• This application is a continuation-in-part (CIP) of Ser. No. 11/248,214, filed on Oct. 13, 2005, pending, and claims benefit of this earlier filing date under 35 U.S.C. §120, the contents of which are herein incorporated by reference to the extent allowed by law.
FIELD OF INVENTION
• The present invention generally relates to an assay system and apparatus involving specific binding of analytes and/or ligands, and specifically relates to chromatographic flow binding assays with a colored conjugate, and a novel lateral-flow-platform that includes a dried or lyophilized colloidal sphere conjugate, such as gold, to the back of the lateral flow test.
BACKGROUND OF THE INVENTION
• It is increasingly desirable to provide a rapid high sensitivity system to detect low levels of ligands in body fluids, plant extracts, environmental samples, tissue samples and enrichment broths. Ideally, such systems should have a minimal number of procedural steps and yield reliable results, even when used by untrained persons. A ligand may be a specific sequence of amino acids or molecule found on proteins such as an antibody, protein receptors, bacterial/microbial peptides, hormone, or drug that binds to a receptor. A receptor is any of various specific protein molecules in surface membranes of cells and organelles to which complementary molecules, such as hormones, neurotransmitters, antigens, or antibodies, may become bound. The ligand may also be a chemical intermediate or reactant with an analyte. Analytes are substances that bind to a ligand. Ligands or analytes may also be peptides, drugs, carbohydrates, haptens, chemicals, chemical reaction with an intermediate compound, and the like.
• To a significant extent, many known tests presently available for detecting ligands are either time consuming, labor intensive, or in need of instrumental assistance to read results. Most known tests also lack an acceptable degree of sensitivity or specificity. This is unfortunate since rapid testing is important for diagnosis and treatment of various physiological conditions; detection of certain strains of micro-organisms; determination of the most appropriate antibiotic treatment of a patient; detection of drug analytes in individuals; detection of cancer cells in a patient's bio-fluid; detection of antibody to a microbial agent; detection of a disease-state protein; and the like.
• Although known types of ligand-receptor assays have been used to detect the presence of various substances, such as ligands, there is a need in the art to provide a rapid, high sensitivity assay requiring a minimum degree of skill from a user. Rapid test assay devices for field use, such as in a home or doctor's office are known in the art for detecting proteins, peptides, drugs, carbohydrates, haptens, chemicals, chemical reaction with intermediate compounds, and the like. Such devices are referred to as one-step lateral flow or one-step immuno-chromatographic assays. These types of assay devices require a minimal number of steps and can be performed by an untrained person. Examples of these test devices abound, and the individual components of a typical one-step lateral flow test are described below:
• A first type of test device involves a test strip of rectangular or square dimensions made of a vinyl, polypropylene, or other pliable or non-pliable plastic laminate to serve as a backing to hold in place other test components that are on an adhesive bond on the backing. A ligand, protein or analyte binding membrane having discreet zones of immuno-reactive proteins or substances immobilized or attached, are usually in linear impregnation or spotted through a dropper onto the membrane. The membrane may be composed of nylon, nitrocellulose, mixed cellulose esters, polysulfones, and the like. If the assay is used to detect an animal antibody, the immuno-reactive protein coated on the membrane may be a ligand to which antibody contained in a positive sample reacts. These immuno-reactive proteins used to coat the membrane are typically native or recombinant proteins derived from Human Immunodeficiency Virus (HIV), human T-cell lymphotropic viruses (HTLV), Mycobacterium tuberculosis (TB), and the like. In a second construct, the membrane may also be impregnated with anti-antibody to capture total class and subclass immunoglobulins. In this case, the specific antibody in the sample may react with native or recombinant ligand proteins, such as HIV, HTLV, or TB, coated onto the surface of the conjugate particles. A third construct may have native or recombinant protein antigens coated on both conjugate and membrane surfaces that react with separate binding domains on the antibody. If the membrane is detecting antigen as the analyte, the surface may be impregnated with antibody or ligand reactive with the antigen. Constructs one, two and three are known as “sandwich” type rapid assays, since the analyte being detected is captured (sandwiched in between) by both the immuno-conjugate and the membrane surface. A fourth method is a competitive inhibition assay, wherein sample analyte at detectable levels either saturates the anti-analyte conjugate or saturates the immobilized anti-analyte capture line on the membrane resulting in no visible line formation. A negative reaction in this case results in a visible line formation, due to the binding of the conjugate with the test line.
• A second group of test devices involve a fibrous membrane, such as, for example, glass, polyester, cotton, or spun polyethylene, in contact with a membrane containing ligand and bound to densely colored particles such as latex, gold, silver, selenium, carbon, and the like. The bound ligand is complementary to the assay being constructed and reacts with the analyte being detected. The coated colored particles are often described as an immuno-conjugate. Sufficient molecules of ligand are coated onto the surface of the colored particles so that when a positive reaction does occur the discreet, striped, or spotted zones on the membrane surface are visible to the naked eye. A sample negative for the ligand being detected may leave a white zone in a sandwich type immuno-assay. If the assay is a competitive inhibition type, a negative sample yields a visible line or spot. The colored particles may be dried down onto the fibrous pad or membrane and placed at the dorsal end (at the opposite end of the absorbent pad or membrane) of the membrane. Release agents may be contained in the dried down colored particles to facilitate re-hydration of the particles, allowing them to react with the analyte being detected.
• A third group of test devices include a fibrous sample receiving pad or membrane, such as glass, polyester, cotton, or spun polyethylene, that is partially in contact with the immuno-conjugate and serves as a reservoir for absorbing and releasing sample. The sample may contain chemicals to facilitate reactive qualities of the assay. The sample may be any biological fluid (bio-fluid) such as tissue extracts, blood, serum, plasma, tears, perspiration, urine, or saliva. The sample may also be derived from an environmental extract, plant extract, or microbial enrichment broth. When a sample or diluted sample is applied to the sample receiving pad or membrane, the movement of liquid is chromatographic and unidirectional towards the absorbent pad or membrane. During migration, the sample re-hydrates the colored particles and reacts with ligand bound to the particles.
• Other test devices are known. For example, one has a bulbous absorbent pad or membrane, which serves as a reservoir for absorbing all liquid components that pass through the membrane. The absorbent material is typically cotton or paper. Another has a zone applied to the membrane containing a control line indicating sample was applied, or that the assay's immuno-conjugate is functional. The control can react with the sample or conjugate. Still another has buffered diluent often used to dilute and condition the sample being detected, allowing for exotic chemistries to occur, thus improving assay performance. The diluent is composed of salt solutions, detergents, and the like and may be applied from a dropper tip, vials, or is contained in a sample vial. And finally another has a plastic or cardboard housing to contain, provide support, and allow ease of use for the whole strip.
• It is important to note that in the known prior art the conjugate pad or membrane is in the same plane as the membrane, and overlaps, or is in constant contact with the membrane. Further, the conjugate is in dry form disposed or deposited in the flow path upstream of the test site. This gives rise to a very specific flow path wherein the sample rehydrates the dried conjugate and travels through the absorbent pad or membrane. The flow path is known as an absorbent flow path since the liquid sample is absorbed by the sample receiving pad or membrane, and next wets the conjugate pad or membrane. Typically, the amount of sample required to wet the conjugate pad or membrane is about 10 to 20 microliters (μl). The sample and conjugate mixture subsequently travels through the conjugate pad or membrane, then contacts the membrane and flows onto the test (flow) membrane.
• In use, in most lateral flow assays using antigen-antibody reactions, the immuno conjugate is composed of synthetic conjugates having latex microparticles, enzymatic, fluorescent, or visually observable metal sol tags. In all these assays, there is a receptor, for example an antibody, which is specific for the selected ligand or antigen, a means for detecting the presence, and often a quantity of the ligand-receptor reaction product. Examples of such qualitative assays include blood typing and most types of urinalysis. For these semi-quantitative type positive/negative tests, visually observable indicia such, as the presence of agglutination or a color change are preferred.
• Positive/negative assays (or semi-quantitative) must still be very sensitive because of the often small concentrations of the ligands of interest in the test fluid. False positives may be troublesome, particularly with agglutination and other rapid detection methods such as dipstick and color change tests. Because of these problems, sandwich and competitive inhibition assays and other detection methods that use metal sols or other types of colored particles have been developed in an attempt to increase sensitivity of detection. In addition, the use of direct labels attached to one of the specific binding ligands produces instant analytical results without the need to add further reagents. Nevertheless, these techniques have not solved all problems related to sensitivity, neither have they been reliable in obtaining results with a minimal number of method steps encountered in these rapid detection methods.
• Various other techniques have been devised for labeling one member of a specific binding pair so that the binding reaction may be indirectly observed. The results of specific binding reactions are generally not directly observable. Useful labels include radiolabels, chromophores, fluorophores, (the presence of which may be detected by means of radiation detectors), spectrophotometers, the naked eye, and the like. Where members of a specific binding pair are tagged with an enzyme label, their presence may be detected by the enzymatic activation of a reaction system including a signal generating a substrate/cofactor group wherein a compound, such as a dye, is activated to produce a detectable signal.
• Other specific binding assay devices are known in the art having vertically arranged elements including the following: a porous capture material impregnated at a reaction site with a member of a specific binding pair such as an antibody or an antigen; a removable prefilter disposed above the capture material; and, a blotter disposed below the capture material. A sample liquid such as blood, serum or other bio-fluid is added to the device wherein the prefilter removes particulates and other impurities from the sample. These impurities would otherwise be trapped on top of the specific binding capture material. Analyte substances within the sample are trapped by means of specific binding reactions with their specific binding partners on the capture material. Non-analyte components of the sample solution pass through the capture material and are absorbed by the blotter. Wash steps may be carried out to remove non-analyte components from the capture material and additional reagents such as enzyme substrates, cofactors and dye precursors may be added to the capture material to indicate the presence or absence of analyte at the reaction site. The prefilter should then be removed so that the presence or absence of analyte at the reaction site may be visually determined. Unfortunately, while these devices are somewhat useful, they suffer from limitations in capture efficiency and sensitivity because most of the analyte in a sample flows around or through the reaction site on the capture material.
• Several one-step lateral flow immunoassay devices, having a strip capable of transporting a developing liquid by capillary action having a first zone for receiving a sample, a second zone impregnated with a first reagent capable of being transported by the developing liquid, and a third zone impregnated with a second reagent, are known in the art (See generally, U.S. Pat. Nos. 4,366,241; 4,094,647; 4,235,601; 4,361,537; and 6,841,159). U.S. Pat. Nos. 6,352,862; 5,622,871; and 5,120,643 also relate to one-step lateral flow tests wherein the conjugate is in dry form along the flow path.
• A major disadvantage of all of the known prior art is that the sample rehydrates the dried conjugate along the flow path. Thus, the majority of sample applied is not involved in the immunological reaction. For example, between 10 μl to 20 μl of sample is all that is required to fully rehydrate a 5 by 5 mm gold pad or membrane with the conjugate pad or membrane composed of polyester, glass, nylon, cellulose or other fibers. Since the conjugate pad or membrane is in the device flow path and in contact with the membrane, a minimal remaining sample is involved in the immunological reaction. The remaining 50 μl to 150 μl of sample acts as a liquid front by pushing the reacted analyte conjugate complexes through the flow path. Thus, a severe limitation of the one-step lateral flow assay is the limited amount of sample actually involved in the reaction with the conjugate. None of the known prior art teach or suggest locating the conjugate pad or membrane on the reverse side of the strip, and not in contact with either the sample receiving pad or the membrane. By locating the conjugate pad or membrane on the reverse side of the membrane, the entire sample first rehydrates the conjugate resulting in a uniform dispersion of conjugate through the entire applied sample. Further, in a one-step lateral flow assay, as a sample mixes with a dry conjugate, incomplete mixing often yields conjugate particles with vastly different numbers of captured analyte molecules. This alone may be a source of decreased sensitivity since the maximum number of analyte molecules are not captured on the surface of the conjugate particles.
• Other designs are known in the art also include two-step assays. These often require the conjugate pad or membrane to be physically and spacially removed from the test strip. For example, there are assays known in the art where the conjugate is contained in book form, or a “male to female” molded apparatus. In the book form, the test strip is located on one panel of the book while the conjugate is located on the other panel. The test requires the user to close both halves for the test to begin. With the “male to female” apparatus, the sample is applied to the male portion of the apparatus, and the two halves are closed to initiate the reaction. Another format has the conjugate dried and located on a separate sampling stick. The sampling stick is mixed with a sample and is then fixed in the cassette sample well. For example, some two step assay platforms are known in the art, such as indicated in U.S. Pat. No. 6,824,997 and U.S. Pat. No. 5,418,171. These tests have a significant drawback though. The end user must perform an additional step to physically bring the membrane and conjugate into contact after the sample has been adequately mixed.
• Thus, there is a desire and need in the art for a high sensitivity assay system and apparatus to rapidly detect low levels of ligands in a small sample size of fluid. Such new tests should involve a minimal number of procedural steps while at the same time, yielding reliable results, even when used by untrained persons. Such a system and apparatus would be useful in areas of infectious disease, pregnancy, microbial detection, ovulation, cancer marker identification, autoimmunity, cardiac markers, biowarfare agents, allergy, drugs of abuse and environmental monitoring, and the like.
SUMMARY OF THE INVENTION
• The present invention generally relates to a rapid high sensitivity system apparatus and method for detecting low levels of ligands in body bio-fluids, environmental and tissue culture extracts, with high sensitivity and specificity.
• The invention includes a system and method for a semi-qualitative (e.g., positive/negative) indicator, such as the presence of agglutination or a color change, as well as quantitative determination. Accordingly, it is an important aspect of one embodiment of the present invention to provide a system and method of analyte detection having a membrane strip that increases detection on the order of 2 to 10 fold over the conventional chromatographic specific binding assay techniques by placing a dried or lyophilized colloidal sphere conjugate.
• Another aspect of the invention includes the placement of the sample port on the same side of the test cassette as the visualization window for simplicity and convenience. The laminating material over the sample pad or membrane serves as a temporary obstruction or impedes in the flow path of a sample solution, resulting in the sample and conjugate label to be in contact for a longer amount of time before flowing out of the conjugate pad. This allows for a more thorough or complete mixing of the conjugate and sample solution before it enters the sample pad or membrane. Preferably gold colloidal spheres are used as conjugates, but other metal sols and latex microparticles may be used as well.
• Another important aspect of the invention is the inclusion of a novel conjugate pad or membrane solution dried onto the conjugate pad or membrane. This solution is composed of negatively charged detergents that are coated onto the conjugate pad or membrane. When sample contacts a conjugate pad or membrane, the labeled colored particles are rapidly expressed into the sample where the test ligand has an increased opportunity to react with said conjugate particles.
• Other important aspects of the present invention may include several cassettes for use with the test strip. The cassettes are designed to allow for sample loading either on the reverse of the cassette, the cassette side, or on the front of the cassette. When loading the sample on the front or side of the cassette, the cassette contains ridges or channels designed both to transport the sample directly to the conjugate pad or membrane and to pre-mix the sample and conjugate.
• Additional aspects and advantages of the invention will become apparent from the following detailed description, the drawings, and the appended claims.
BRIEF DESCRIPTION OF THE FIGURES
• The foregoing features, as well as other features, will become apparent with reference to the description and figures below, in which like numerals represent like elements, and in which:
• FIG. 1 is an expanded side view of chromatographic elements assembled into a test device in accordance with the objects' present invention;
• FIG. 2 is an expanded side view of prior art chromatographic elements assembled into a test device;
• FIGS. 3a-3care side views of chromatographic elements assembled into a test device with alternative layouts of the conjugate pad or membrane isolated from the sample pad or membrane in accordance with the objects' present invention;
• FIGS. 4a-4care side views of chromatographic elements assembled into a test device with alternative layouts of the conjugate pad or membrane in accordance with the objects' present invention;
• FIG. 5 is a perspective view of a chromatographic assay device enclosed in a chamber in accordance with the objects' present invention; and
• FIG. 6 is an expanded side view of a cassette holder in accordance with the objects' present invention.
• FIG. 7 is an example of a cassette configuration of the present invention adding detail showing homogeneous mixing.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
• The present invention generally relates to a rapid, high sensitivity chromatographic assay for detecting low levels of ligands in bio-fluids, environment, plant and tissue culture extracts, using a minimal number of procedural steps even when used by untrained persons. The present invention encompasses diagnostic kits that may contain a chromatographic specific binding assay system, and preferably an immunochromatographic specific binding assay system. Furthermore, the system and apparatus, because of its accuracy and simple method steps, make it appropriate for field use such as a home, clinic, point of care setting, or doctor's office. Test results may be visually read or read by an instrument known in the art and readily available to give either a semi-qualitative (e.g., a positive/negative result) or a quantitative result. In use, the present invention is simple to use and requires a minimum degree of user skill and involvement.
• To assist in understanding the present invention,FIG. 2 illustrates a side view of an expanded prior art assay platform device having chromatographic elements and is generally indicated at42. The main components ofdevice42 are a sample receiving pad ormembrane13 composed of a fibrous membrane such as cotton, glass fibers, polyester, nitrocellulose or nylon (where thesample fluid10 is initially applied) and acassette16 shown in expandable halves and a laminate11. Aliquid sample10 is applied through holes (cassette16 openings)16bonto a sample receiving pad ormembrane13. Thesample fluid10 flows out of the sample receiving pad ormembrane13 toward an adjacent dried or lyophilizedmobile conjugate pad15 and is along aflow path20, composed of a fibrous membrane, where thesample10 andmobile conjugate22 has an opportunity to mix and flow through capillary migration into an elongated lateral flow analyte detectionfibrous membrane strip12 toward the direction of reservoir pad ormembrane14, located at the opposite end of the sample receiving pad ormembrane13 conjugate.Sample10 andconjugate mixture22 then continue to migrate to an immobilized capture reagent membrane atregion12aofdetection membrane strip12, where the sample/conjugate mixture can bind or stick to the immobilized capture reagent, causing a color reaction, indicating the presence of a specific ligand or analyte. The presence and/or amount of analyte in thesample10 may be determined by the visibility of a line formed by thecapture reagent12a, specific for the analyte-label reagent conjugate being tested. Detection may be observed through aviewing window area16aofcassette16 having a transparent cover. There is also acontrol reagent region12b, which is used to verify that the reagents are reacting as they should. The membrane strips or pads are attached to laminate11 (semi-rigid) to maintain its elongated structure. Acassette case16 is also used to maintain rigidity and prevent contamination of fluids that may alter the capillary flow of the foreign fluids toward the reservoir pad ormembrane14.Membrane14 is typically composed of cotton, paper or glass fiber.Membrane12 regions may be composed of nitrocellulose, mixed cellulose ester, nylon and the like. A major shortcoming of all of the known prior art is thatsample10 rehydrates the driedconjugate22 along theflow path20 at the conjugate pad ormembrane15, starting from thesample application area16b, flowing in the direction of the reservoir absorbent pad as shown at20 inFIG. 2. Thus, the majority ofsample10 applied is not involved in the immunological reaction. In fact, most of thesample10 acts as a liquid front, pushing the reacted analyte conjugate complexes throughflow path20 and is actually not involved in the reaction with theconjugate22.
• FIG. 1 illustrates a possible embodiment of the present invention assay platform device and is generally indicated at34.Device34 is shown in expanded side view to illustrate the individual component configuration of the chromatographic elements. Noted differences over the prior art are the change in location of cassette opening (sample well)16bto the underside ofcassette16 and its immediate contact with conjugate pad ormembrane15.Device34 may be configured to perform at least four types of assays including: secondary antibody sandwich for measurement of sample antibody; antibody-antigen-antibody sandwich for measurement of antigen in either competitive (inhibition) or non-competitive mode; antigen-antibody-antigen sandwich for measurement of antibody; and competitive inhibition assay involving antigen bound to the conjugate microparticles, anti-antigen on the membrane, with detectable analyte inhibiting the anti-antigen reaction.
• In the proposed immunoassay device of the present invention, the dried or lyophilized conjugate pad ormembrane15 may contain metal sols, enzymatic, fluorescent, latex microparticles, and the like, preferable colloidal gold particles. Conjugate pad ormembrane15 is attached to the side of laminate orsemi-rigid material11, opposite sample receiving pad ormembrane13. Therefore,Laminate11 is semi-rigid and can be configured to be impervious to sample flow or permeable by any number or type of pores, slits, or other types of permeable substances known in the art to functionally slow the capillary effect of the sample flow. The entire elongated assay strip and components are enclosed in aplastic cassette16.Liquid sample10 is applied to holes oropenings16bdirectly unto the conjugate pad ormembrane15, allowing a complete mixing of theconjugate particles22 withsample fluid10. The completelymixed sample10 andconjugate22 next flows by gravity and capillary action around and/or through the laminate11 onto sample receiving pad ormembrane13 alongflow path30. This allowssample fluid10 to rehydrateconjugate particles22, creating a more complete mixture ofsample10 andconjugate22 and remain in contact with each other for an longer amount of time, compared to the application methods in the prior art.Sample10 and mobile conjugate mixture next flows through capillary migration into the elongated lateral flow analyte detectionfibrous membrane strip12 along flow path30 (toward the reservoir pad ormembrane14, located at the opposite end of the sample receiving pad or membrane13).Sample10 andconjugate22 mixture then continues to migrate to an immobilized capturereagent membrane region12a, where the sample/conjugate mixture can bind or stick to the capture reagent, causing a color reaction, indicating the presence of a specific ligand or analyte. The presence and/or amount of analyte in thesample10 may be determined by the visibility of a line formed by thecapture reagent12a, specific for the analyte-label reagent conjugate, which is detected in theviewing window area16awhich may be a transparent cover over an opening in thecassette cover16 and known in the art. There is also acontrol reagent feature12b, which is used to verify that the reagents are reacting as they should. The major advantage of the present invention over the prior art is theflow path30 may now be one in which the liquidphase containing sample10 andconjugate22 is first pre-mixed for an extended amount of time, prior to contacting sample receiving pad ormembrane13. The dried orlyophilized conjugate22 in theconjugate membrane15 may consist of latex microparticles, enzymatic, fluorescent, or visually observable tags such as silver, selenium, carbon, other metal sol tags, preferable colloidal gold spheres. Thedetection membrane strip12, sample receiving pad ormembrane13, conjugate pad ormembrane15, or reservoir pad ormembrane14 may be composed of a series of porous material pieces such as, paper, cotton, polyester, glass, nylon, mixed cellulose esters, spun polyethylene, polysulfones, and the like. Preferably, nitrocellulose, nylon or mixed cellulose esters are used for the analytedetection membrane strip12, while paper, cotton, polyester, glass fiber or polyethylene are preferred for theconjugate pad15, sample receiving pad ormembrane13 andreservoir absorbent14.
• In atest tube40 version of the assay illustrated atFIG. 5 conjugate pad ormembrane15 andsample10 are mixed for an longer amount of time than known is the art, prior to contact with the sample receiving pad ormembrane13 and flows along apath46. Thus, in both thetest tube40 version andcassette16 version the flow path is one in whichsample10 andconjugate22 go through a liquid, non-absorbent phase prior to being absorbed onto a sample receiving pad ormembrane13.
• In one embodiment of the present invention thedetection membrane12 and sample receiving pad ormembrane13 and conjugate pad ormembrane15 may optionally contain blocked bovine serum albumin (BSA) and detergent to prevent loss of human antibody, by non-specific attachment.
• A reservoir pad ormembrane14 at the distal end of the device receives and absorbsliquid sample10 andconjugate22 material to facilitate capillary migration through thedetection membrane12 along the pre-configured flow path. A buffered diluent may be used to dilute andcondition sample10 being detected, allowing for binding and/or chemical reactions to occur to improve assay performance. The diluent may be composed of salt solutions, detergents, and the like and may be applied from a dropper tip vials or contained in a sample vial. Acapture ligand12ais attached to thedetection membrane12, and asecond region12bperforms a built in control feature.
• The whole strip should be contained in a plastic or cardboard housing (cassette16) to provide support and for ease of use. Thecassette16 may contain ridges designed to both mix thesample10 andconjugate22 and to facilitate eventual contact with the sample receiving pad ormembrane13. The cassette may have ridges or channels to facilitate (channeling) ofsample10 to contact the conjugate pad ormembrane15.
• An alternate embodiment of the present invention hassample port16bon the same side of the same plane oftest cassette16 asvisualization window16a. The advantage of this alternate embodiment is simplicity and convenience of the cassette to the user. Thevisualization window16acan be monitored after direct sample application, keeping the test strip in the same physical orientation or position. FIGS.3(a-c) showsample port16bon the same side oftest cassette16 asvisualization window16a.
• FIG. 3ashows a design where sample-conjugate pad15 is located in a displaced position on the back side of laminatingmaterial11. The Figure also shows a hinge-point orregion77, about whichlaminating material11 can be folded to result in the embodiment shown inFIG. 3b. This embodiment placesconjugate pad15 overSample receiving pad13 such that when sample is applied to the conjugate pad, where thelaminating material11 is also over thesample pad13 serving as a temporary obstruction or impedance to the flow path of a sample solution intosample pad13, thereby facilitating or assisting in a more thorough or complete mixing of the conjugate and sample solution before it enterssample pad13. This temporary obstruction breaks down when the combined effects of the sample volume and surface tension cause the sample to run around and/or eventually through laminatingmembrane11. The extended mixing permitted by this temporary obstruction is an important feature of the present invention providing the sample liquid and conjugate label to be in contact with each other for a longer amount of time before flowing intosample pad13. This temporary obstruction by the laminated material can be regulated, or adjusted, by piercing the laminating material, or by using a membrane with micropores. The temporary obstruction can also be accomplished through the use of a slow-wetting membrane, which is not impervious, of any kind between the conjugate pad and the sample pad. The extended mixing permitted by this temporary obstruction is the key, not whether the membrane is impervious or not, providing the sample liquid and conjugate label to be in contact with each for a longer amount of time before flowing into thesample pad13. This results in increased analyte detection sensitivity and is a salient difference over the prior art that gives the 2-step test its superior performance. The temporary flow obstruction provided bylaminate11 can be regulated or adjusted, by: piercing the laminating membrane, using a membrane with micropores, placing varied capillary flow channel membranes of any kind between the conjugate and the sample pad or membrane and the like, known in the art. Thus the laminate or laminating strip function is not identified by being impervious, but is characterized by whether the laminate provides the necessary poricity to provide temporary obstruction of sample fluid intosample membrane13. Over time a sample liquid will flow fromconjugate pad15 and migrate around and/or throughlaminating material11 into thesample pad13.
• FIG. 3cshows a design where thelaminating material11 over the sample pad is separated (i.e., not hinged) as inFIG. 3c). The laminating material in bothFIGS. 3band3ctemporarily isolatesconjugate pad13 fromsample pad15 such that more time is provided for the complete mixing of a sample liquid with the rehydrated conjugate in conjugate pad ormembrane13 before migrating into sample pad ormembrane15, thereby delineating these 2-step designs from prior 1-step lateral-flow assays.
• FIGS.4(a-c) illustrate additional alternative embodiments of the present invention wherein the chromatographic elements assembled into an assay platform device. The location of the conjugate pad ormembrane15 is varied as is the shape oflaminate11 to illustrate different placement locations of the dried or lyophilized mobile conjugate pad ormembrane15. These variations illustrate a key technical advantage of the present invention over the prior art in that it is not dependant upon the exact placement of the dried or lyophilized mobile conjugate pad ormembrane15. Rather the concept of the application of aliquid sample10 directly to the dried or lyophilized mobile conjugate pad ormembrane15, located on the opposite side and upstream of the sample receiving pad ormembrane13, enabling a more complete mixture with the conjugate22 andsample fluid10.
• Applying the present invention provides membrane strips and improved methods for the practice of chromatographic specific binding assay techniques.Detection membrane strip12 is selected to have a sufficient pore size such that the conjugate22 reagent may be comprised of latex, gold, silver, selenium, carbon, but are not limited to these elements.Sample10 migrates by capillary action.Sample10 size is determined by the capacity of thewick pad14 and may be as much as 500 μl compared to 10 μl to 20 μl ofeffective sample10 as constrained by one-step lateral flow assays in the prior art.Test samples10 interact with the conjugate22 to produce a near complete homogeneous mixture ofconjugate22 andsample10. Thus eachconjugate particle22 contains a uniform number of captured analyte molecules, and further eachconjugate particle22 contains about the same number of captured analyte molecules as shown inFIG. 1.
• By placing the conjugate pad ormembrane15 containing ligand bound to densely colored particles such as latex, gold, silver, selenium, carbon, and the like on a reverse or opposite side of thesample receiving membrane13, but not in contact with the sample receiving pad ormembrane13; the analyte detection improves on the order of 2 to 10 fold over the conventional chromatographic specific binding assay techniques. In simplest practice as illustrated inFIG. 5 thetest strip44 is placed intest tube40 containing approximately 110 μl to 500 μl ofsample10. When thesample10 solution contacts the conjugate pad ormembrane15,particle conjugate22 is rapidly expressed into thesample10 where the test ligand has opportunity to react with saidconjugate particles22. Assample10 migrates into the flow membrane, the ligand-particle conjugate is carried alongflow path46 where it is given opportunity to react with the capture ligand attached to thedetection membrane12.
• Thedetection membrane strip12 of this invention is characterized by a high capture efficiency, which is particularly advantageous in the use of the invention for diagnosis of pediatric and geriatric patients where the volume ofsample10 fluids obtainable from such patients may be limited.
• This lateral flow assay platform is also suitable for analysis ofsamples10 with heavy loads of particulate matter without the necessity of a prefilter. Particulate matter does not interfere with analyte determination at the location of thereaction site12a, but instead accumulates at the interface of the sample application means and the chromatographic material. Nevertheless, prefilters, may be used and fitted into sample application means for samples having especially heavy loads of particulate matter, for example, whole blood.
• FIG. 6 shows a diagram of an assembled strip holder (cassette16).FIG. 6ashows the viewing side ofcassette16 and correspondinglaminate11 configuration.FIG. 6bshows a view of thecassette16 having asample receiving opening16band the corresponding view oflaminate11. This apparatus performs two functions, first thesample10 andconjugate22 mix to homogeneity, and second, the mixture flows through thedetection membrane12, for detection at theviewing window16a.Cassette16 ortest tube40 is critical to proper mixing of the conjugate andsample10. This also is distinguishable over the one-step lateral flow assay in thatcassette16 ortest tube40 is required for theentire sample10 to mix with the dried conjugate at conjugate pad ormembrane15.
• Test Tube40 Version of the Invention
• One embodiment of the present invention allows the chromatographic test assay to be performed in atest tube40 as shown inFIG. 5. In this case,sample10 is added totube40 anddetection strip44 is inserted. The properties of the conjugate22 are such that it immediately goes into solution upon contact withsample10.Conjugate22 andsample10 come to homogeneity rapidly, and the mixture then flows to the sample receiving pad ormembrane13 and analysis of thedetection window16ais performed.
• Cassette16 Version of the Invention
• In another embodiment asample10 is applied on the reverse side ofdetection strip12 via anopening16bin thestrip cassette16. Assample10 mixes the conjugate22 in the conjugate pad ormembrane15, it quickly collects in the bottom ofcassette16. Further,cassette16 may contain plastic rods, chambers, or channels which further allow for a homogeneous mixing of the conjugate22 andsample10 as shown inFIG. 6. The conjugate-sample mixture may be moved to thedetection membrane strip12 via channels orridges48 located in the cassette as shown inFIG. 6.
• In one embodiment of the present invention, theassay strip34 has detecting antibodies. The immuno-reactive protein may be a ligand to which antibodies contained in apositive sample10 reacts with such as HIV, HTLV, TB, and the like.Detection membrane strip12 may be impregnated with anti-antibody to capture total, class and subclass immunoglobulins. In this case, the specific antibody insample10 reacts with ligand peptides (HIV, HTLV, TB, and the like) coated onto the surface of theparticles22.
• In another embodiment of this invention,device34 may have antigen on both the conjugate pad andmembrane15 anddetection membrane strip12 surfaces to which specific antibody may then be able to react with separate binding domains on the antibody. Ifdetection membrane strip12 is detecting antigen as the analyte, themembrane12 surface may be impregnated with antibody or ligand reactive with the antigen. Typical antigen capture examples are specific antigen peptides, HIV p24 antigen, Hepatitis B surface antigen, cardiac markers, bacterial cells and the like and are known in the art. A colored line formed in this area may indicate detectable levels of analyte. A control zone, which indicates to the user whether or not the test was successfully run may also be included and illustrated in figures. In this example, the control inregion12breacts with the conjugate. Often for human samples the control line is an antibody fragment, such as Fragment antigen binding (FAB)′2 goat anti human IgG (H&L) from Jackson Immunoresearch or KPL. (FAB)′2 has two antigen-binding arms that remain linked. Using FAB material prevents the binding of the protein A coatedgold particles22, and indicates the addition of human sample10 (containing antibody). If buffer alone is added to the test strip, no control line develops resulting in an invalid assay. A colored conjugate to which purified proteins, haptens, or antibody to human immunoglobulins is chemically attached. The dried orlyophilized conjugate22 on the conjugate pad orconjugate membrane15 is mixed with special solubilizing and releasing compounds in a buffer; facilitating any interaction with thesample10 bio-fluid.
• The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the specification is hereby incorporated herein by reference.
• While the invention has been described in conjunction with specific embodiments, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, the present invention attempts to embrace all such alternatives, modifications and variations that fall within the spirit and scope of the appended claims.

Claims (20)

1. A chromatographic specific binding assay strip device, comprising:

a laminate strip having a first side and a second side;

a sample receiving pad, a fibrous reservoir pad disposed on said first side of said laminate strip; and a fibrous detection pad disposed between said fibrous sample pad, wherein said fibrous reservoir pad is disposed at the distal end of said device facilitating unidirectional capillary migration through the detection pad or membrane;

a fibrous conjugate pad disposed on said second side of said laminate;

wherein said laminate strip is configured to fold back at a hinge point, such that laminate strip and conjugate pad on the second side are positioned on top of the sample receiving pad;

wherein said laminating strip separates the conjugate pad from the sample pad such that the flow path of liquid migration is slowed.

2. The device ofclaim 1, wherein the impeding or obstructing property of said laminate strip is regulated or adjusted by piercing the laminating membrane, or by using a membrane with micropores.

3. A chromatographic specific binding assay strip device, comprising:

two laminate strips;

a sample receiving pad, a fibrous reservoir pad disposed on said first laminate strip; and

a fibrous detection pad disposed between said fibrous sample pad, wherein said fibrous reservoir pad is disposed at the distal end of said device facilitating unidirectional capillary migration through the detection pad or membrane;

a fibrous conjugate pad disposed on said second laminate strip;

wherein said conjugate pad on the second laminate strip is positioned on top of the sample receiving pad;

wherein said second laminating strip separates the conjugate pad from the sample pad such that the flow path of liquid migration is slowed.

4. The device ofclaim 3, wherein said liquid migration is regulated or adjusted by piercing the laminating membrane, or by using a laminate with micropores.

5. The device ofclaim 1, comprising a cassette housing further comprising a sample well or opening for receiving the liquid sample or test tube version.

6. The device ofclaim 5, wherein the sample port is on the same side of the same plane of the test cassette as the visualization window.

7. The device ofclaim 1, wherein said device contains blocked bovine serum albumin (BSA) and detergent for preventing loss of antibody, by non-specific attachment.

8. The device ofclaim 1, wherein said binding assay strip is an immunological binding assay strip.

9. The device ofclaim 5, wherein said cassette contains ridges or channels designed to both pre-mix the sample with conjugate, and facilitates a unidirectional flow path.

10. The device ofclaim 1, wherein a pre-filter is fitted into a sample application means to remove heavy loads of a particulate matter.

11. A kit comprised of the device ofclaim 1, further comprising a buffered diluent and wherein said conjugate is a colloidal metal.

12. A kit comprised of the device ofclaim 5, further comprising a buffered diluent and wherein said conjugate is a colloidal metal.

13. A method of detecting the presence or absence of an analyte or ligand in a liquid sample comprising the steps of:

application of a sample fluid directly unto a dried or lyophilized conjugate in the conjugate pad or membrane region ofclaim 1, wherein said sample fluid rehydrates the conjugate pad or membrane, producing a uniform dispersion of conjugate through the entire applied sample fluid creating a sample-conjugate complex, prior to entering the sample receiving pad or membrane ofclaim 1;

allowing the sample and conjugate mixture to flow by capillary action into the sample receiving pad or membrane, permitting said sample conjugate mixture to flow unidirectionally into the lateral flow analyte detection membrane in the direction toward the reservoir pad or membrane; and

detecting the presence or absence of an analyte or ligand in the detection viewing region containing the capture reagent capable of binding the sample-conjugate complex and a control reagent region, both capable of color reactions.

14. The method ofclaim 13, wherein the analyte or ligand is selected from the group consisting of proteins, modified proteins, hormones, peptides, drugs, carbohydrates, haptens, chemicals, and chemical reaction with an analyte.

15. The method ofclaim 13, wherein the liquid sample is selected from the group consisting of a bio-fluid, plant extracts, environmental samples, tissue samples and enrichment broths.

16. The method ofclaim 13, wherein detecting the presence of an analyte or ligand is used as an indicator for the presence of an infectious disease, pregnancy, microbial detection, ovulation, cancer marker identification, autoimmunity, cardiac markers, biowarfare agents, allergy, drugs of abuse or environmental monitoring.

17. The method ofclaim 15, wherein the bio-fluid is selected from the group consisting of tissue extracts, blood, serum, plasma, tears, perspiration, urine, and saliva.

18. The method ofclaim 13, wherein said detection is measured by binding selected from the group consisting of:

secondary antibody sandwich for measurement of sample antibody; antibody-antigen-antibody sandwich for measurement of antigen in either competitive (inhibition) or non-competitive mode;

antigen-antibody-antigen sandwich for measurement of antibody; and competitive inhibition assay involving antigen bound to the conjugate microparticles, anti-antigen on the membrane, with detectable analyte inhibiting the anti-antigen reaction.

19. The method ofclaim 13, wherein said conjugate is a colloidal metal.

20. The method ofclaim 19, wherein said colloidal metal is gold.

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