WO2001018235A2

WO2001018235A2 - Lateral flow device with metal oxide indicator and assay method employing same

Info

Publication number: WO2001018235A2
Application number: PCT/US2000/040859
Authority: WO
Inventors: L. Allen Ross; Raymond L. Miller; Timothy J. Barder
Original assignee: Eichrom Technologies, Inc.
Priority date: 1999-09-09
Filing date: 2000-09-08
Publication date: 2001-03-15
Also published as: WO2001018235A3; WO2001018235A8

Abstract

A lateral flow device with an indicator comprising a metal oxide label grafted to an analyzing moiety and assay method employing same are disclosed. The invention employs a plurality of metal oxide particles or metal oxide-coated non-porous silica particles as labels grafted to analyzing moieties that selectively bind the analyte, and identify the analyte to which the assay is directed. In a contemplated assay, after a sample is introduced, the analyzing moiety selectively binds the analyte, if present, to form an indicator-analyte complex. The complex flows through the device via capillary action to the receptor region where a second, capture, moiety that is affixed to the assay medium binds the complex thereby causing metal oxide indicators to accumulate. The assay can then be interpreted by a characteristic property of metal oxide label such as color at the receptor region.

Description

LATERAL FLOW DEVICE WITH METAL OXIDE INDICATOR AND ASSAY METHOD EMPLOYING SAME

Description

Technical Field

The present invention relates to a lateral flow device and assay method, and more particularly, to a diagnostic test kit that employs metal oxide or metal oxide-coated particles grafted to analyzing moieties as indicators.

Background Of The Invention

Lateral flow or immunochromatographic color change devices have become the preferred method for rapid diagnostic tests. Their simplicity and ease of use combined with the plethora of available antibodies and other binding moieties and readily automated manufacturing procedures have provided for a broad acceptance of the technology. Such devices commonly are used in several diagnostic applications such as, for example, home pregnancy test kits and other simple diagnostic tests.

In a typical assay, the lateral flow device employs an indicator, also sometimes referred to as a conjugate, comprising a selectively reactive analyzing moiety with a label grafted thereto. The label has some characteristic, such as color, which is used to identify the presence in the test sample of the particular analyte to which the assay is directed. The analyzing moiety is a binding partner for the analyte. When a liquid sample is introduced and brought in contact with the indicator, the analyzing moiety binds the analyte if present in the sample to form an indicator-analyte complex. The complex is then delivered via capillary action to a second selectively reactive moiety or "capture moiety" , which is fixedly attached to a downstream surface of the device. The second moiety binds the complex at a second site thereby capturing the complex in what is commonly referred to as a "sandwich" . As multiple complexes reach the capture site, they accumulate there. The presence of the analyte can then be identified and interpreted based on the accumulation of color (or some other characteristic property of the indicator label) at the capture site.

Common analytes and moieties include ligands such as antigens, antibodies, deoxyribonucleic acid (DNA) , ribonucleic acid (RNA) and other substances of either natural or synthetic origin that are of diagnostic interest and have a specific binding partner therefore.

The term "antibody" as used herein and in the art in general refers to a molecule that is a member of a family of glycosylated proteins ( "glycoproteins" ) called immunoglobulins that can specifically combine with (bind to) an antigen. Such an antibody combines with its antigen by a specific immunologic binding interaction between the antigenic determinant of the antigen and the antibody combining site of the antibody. The word "antigen" has been used historically to designate an entity that is bound by an antibody, and also to designate the entity that induces the production of the antibody. More current usage limits the meaning of antigen to that entity bound by an antibody, whereas the word "immunogen" is used for the entity that induces antibody production. Where an entity discussed herein is both immunogenic and antigenic, it will generally be termed an antigen.

The analyzing moiety and capture moiety generally comprise the specific binding partner or partners for the analyte . Depending on the type of analyte that is employed, the analyzing and capture moieties are the same or different. For example, in a typical sandwich assay, the analyte is an antigen (e.g., E. coli 0157 :H7) and the analyzing moiety is an anti-E. coli 0157 :H7 antibody that selectively reacts with a binding site antigen on the cell . The capture moiety also is an antibody that selectively reacts with the same or a different antigenic binding site on the E. coli 0157 :H7 cell.

Several kinds of indicators have been used with lateral flow devices, including moieties labeled with colored latex or polymeric particles, often referred to as "beads", or colloidal gold particles. Colored bead technology has progressed substantially over the years with the development of improved polymerization techniques for latex beads and more recently with the advent of gold nanoparticle bead technology. Other known indicators include moieties- labeled with magnetic particles that react with an analyte and then are magnetically drawn to a receptor area for magnetometric identification. Fluorescent labels indicators also have been used, as for example fluorescein isothiocyanate . Radiation labels also are known in the art, as for example gamma ray emitting elements such as ¹²³I, ^{1 5}I, ¹²⁶I, ¹²⁸I, ¹³¹I, ¹³1, and ⁵¹Cr. Positron emitting elements such as ^X1F, ¹⁸F, ¹⁵0 and ¹³N also have been used.

The properties of the indicator are critical for successful development of a lateral flow device. The particle size and distribution, hydrophobic properties, flow characteristics and stability of the conjugate are all important to preparing a reproducible test.

Colored latex or polymeric bead indicators have proven to be very successful in binding antibodies and other moieties for numerous assays and can be made with many different moiety-label linking elements or "linkers" .

Colloidal gold provides a unique colored bead matrix that is small enough to work well in typical lateral flow membranes. In a colloidal gold- indicated pregnancy test, for example, antibodies to human chorionic gonadotrophin (HCG) , a hormone released by pregnant women are labeled with gold micro or nano particles. When mixed with a urine sample containing the HCG hormone, the antibodies bind to the hormone and cause the gold particles to coagglutinate, resulting in an accumulation of pink color, indicating the presence of the hormone and a positive pregnancy test. Colloidal gold also has been used for colorimetric DNA detection, as for example is described in Nanomaterials in Analytical Chemistry, Analytical Chemistry News & Features, May 1, 1998 at 323A-324A.

General descriptions of the state of the art in assay devices are contained in U.S. Patent No. 5,922,615 (Nowakowski et al . ) . Additional background regarding the art is contained in U.S. Patent No. 5,622,871 (May et al . ) , U.S. Patent No. 5,591,645 (Rosenstein) , U.S. Patent No. 4,820,505 (Ginsberg et al.) and U.S. Patent No. 4,703,017 (Campbell et al . ) . The disclosures of each of these patents are incorporated by reference.

Each of the above-described indicators has certain drawbacks and limitations, however.

Colored latex or polymeric bead indicators have been difficult to adapt to lateral flow devices because of their relatively large size and high hydrophobic character. Both of these properties make flow optimization difficult. The polymeric matrix also imparts a high degree of nonspecific adsorption. Colloidal gold indicators require surface activation followed by the addition of sulfhydryl- or mercapto- linkers that provide the ability of colloidal gold to conjugate with a wide range of antibodies. The preparation of the conjugate is also somewhat difficult in that the colloidal gold-moiety conjugate is known to be unstable.

Fluorescent indicators generally require ultraviolet radiation and/or other interpretative equipment. There also is the potential for analyte absorption of the fluorescent signal.

The magnetic indicators generally employ a magnetic or paramagnetic label, which requires a magnetometer or similar interpretative device to determine the presence of the analyte.

Radioactive indicators require radioactivity interpretative equipment and impose safety and disposal complications, which add to the expense of the assay and limit the ability to employ such a test outside a controlled environment.

Thus, the indicators used previously in lateral flow devises exhibit one or more limitations or drawbacks in their use. The disclosure that follows provides a lateral flow devise whose indicator avoids many of those limitations and drawbacks to provide a solution to the problems inherent in the prior devices.

Brief Summary Of The Invention

The present invention is directed to a simple, inexpensive, yet highly sensitive assay method and improved lateral flow device that can be used in conjunction with a variety of analytes . The enhanced sensitivity and improvement in the lateral flow device stems from the use of an indicator comprising a metal oxide or metal oxide-coated particle chemically or physically bonded to an analyzing moiety.

Metal oxides are capable of having a variety of identifying characteristics, including visible color, fluorescence, phosphorescence, radioactivity, reflectance, refractive index and magnetism, which make them particularly diverse and versatile indicators. Depending on the metal oxide, any of these properties or some combination thereof can be used to indicate the presence of an analyte in a given sample. For example, a magnetic (magnet responsive) metal oxide particle can be coated with a fluorescent metal oxide layer to permit both magnetometric and fluorescent identification. Such combinations of properties imposed through multiple metal oxide layers also permit for increased device sensitivity and accuracy. Alternatively, the same metal oxide particle or metal oxide-coated particle can have multiple identifying characteristics as in the case of one form of iron oxide, which has a black or black-green color that is useful as a colorimetric indicator and also is magnetic (magnet responsive) . In a contemplated assay, the colorimetric identifying characteristic can be used for an initial test prognosis, which can be confirmed through the use of a magnetometric test .

Metal oxides also have readily functionalizeable surfaces and have been found to be excellent indicator hosts for a variety of protein/ligand based moieties.

An additional benefit of metal oxide indicators is their hydrophilicity. Thus, when an aqueous sample is introduced to a testing medium, the metal oxide or metal oxide-coated particles flow freely through the testing medium and deliver the analyte, if present, to a receptacle portion disposed thereon for identification. The easy flow of the metal oxide and metal oxide-coated particles also reduces the occurrence of non-specific adsorption of analyte to the testing medium.

Metal oxides also permit testing in a wide range of pH conditions, which provides for a broader range of testing environments than, for example, colloidal gold.

In a contemplated assay, after a sample is introduced the indicator comprising a metal oxide label grafted to an analyzing moiety that specifically binds the analyte if present in the sample to form an indicator-analyte complex. The complex flows through the device via capillary action to the receptor region where a second or capture moiety that is affixed to the assay medium binds the complex. The assay can then be interpreted to determine whether the analyte is present, which is indicated by the presence of an identifying characteristic of the metal oxide at the receptor region.

More particularly, the invention contemplates a lateral flow device that comprises an assay medium having a first portion and a second portion in flow communication with each other. The first portion has a site for application of a sample and an indicator comprising a metal oxide particle label grafted to an analyzing moiety. The analyzing moieties specifically bind an analyte when a sample containing the analyte is contacted with the first portion of the assay medium to form an indicator- analyte complex. The complex flows with either liquid from aqueous sample or with an exogenously supplied liquid, as where an anhydrous sample is used, from the first portion of the assay medium to the second portion of the assay medium. A capture moiety affixed to the second portion binds the complex thereby "capturing" the complex. The presence of analyte can then be determined by the particular identifying characteristic of the metal oxide label .

A lateral flow assay method also is contemplated. That method comprises the following steps. A lateral flow device as described above is provided. The lateral flow device is contacted with an aqueous sample to form an indicator-analyte complex when the analyte is present in said sample. The liquid delivers the complex to the capture moiety by capillary action. The complex so delivered is bound by the capture moiety where a population of indicators accumulate. The presence of the analyte is determined by a characteristic property of the metal oxide labels accumulated at the capture moiety.

Brief Description Of The Drawings

In the drawings forming a portion of this disclosure,

FIG. 1 is a top plan view of a lateral flow device employing the moiety-labeled colored metal oxide indicator;

FIG. 2 is a top plan view of a preferred embodiment of the lateral flow device;

FIG. 3 is a cross-sectional view of the lateral flow device shown in FIG. 2 taken along line 3-3;

FIG. 4 is a comparative plot of colony population versus relative intensity for E. coli 0157 :H7 (vertical lines), 0157 :H38 (diagonal lines) and 0157 :H45 (dots) with a metal oxide indicator;

FIG. 5 is a comparative plot of colony population versus relative intensity for E. coli 0157 :H7 (vertical lines), 0157 :H38 (diagonal lines) and 0157 :H45 (dots) with a colloidal gold indicator;

FIG. 6 is a chart depicting the sensitivity of Metal Oxide Indicators for E. coli 0157 :H7; FIG. 7 is a chart comparing a contemplated

Metal Oxide Indicator E. coli 0157 :H7 Test with other polyclonal tests; and

FIG. 8 is a chart comparing a contemplated Metal Oxide Indicator E. coli 0157 :H7 Test with commercial monoclonal test.

Detailed Description Of Preferred Embodiments

The present invention contemplates an assay indicator comprising a metal oxide label grafted (chemically or physically bonded) to an analyzing moiety that specifically binds an analyte when present in a sample. The metal oxide label comprises a colloidal suspension of metal oxide particles or metal oxide-coated non-porous silica particles having a diameter of about 0.1 μm to about 10 μm.

Referring now to FIG. 1, it is seen that in one embodiment, the indicator is used in a lateral flow device 1 that comprises an assay medium having a first portion 3 and a second portion 5 in flow communication with each other.

The first portion 3 has a site 11 for application of an aqueous sample and an indicator 13, comprising an analyzing moiety with a metal oxide label grafted thereto, disposed thereon. The second portion 5 has a capture moiety 7 affixed thereto.

The analyzing moiety of the first portion specifically bind an analyte when an aqueous sample containing the analyte is contacted with the first portion 3 of the assay medium to form an indicator- analyte complex labeled with metal oxide particles. The complex flows with the liquid of the sample from the first portion of the assay medium 3 to the second portion of the assay medium 5 wherein the capture moiety 7 binds to the complex to provide a signal in the form of the particular identifying characteristic of the metal oxide when the analyte is present in the aqueous sample.

I . Preparation of a Preferred Embodiment

During preparation, metal oxide particles, in a colloidal suspension, are introduced to any of a variety of analyzing moieties (receptors) . Such a suspension is often referred to as a "ferro-fluid" where the particles are iron oxide. Preferably these metal oxide particles are on the order of 100 nm in diameter or less. However, larger particles on the order of 200 nm or larger can also be used. These particles, which generally range in size from less than 100 nanometers to about 10 micrometers are hereinafter referred to as metal oxide microparticles . Colloidal metal oxide particles are preferred. Alternatively, particles, preferably silica microspheres having a diameter of about 0.1 μm to about 10 μm, coated with a metal oxide can be used as indicators .

It also is preferred to use the colorimetric properties of the metal oxide as the identifying characteristic of the indicator. Thus, iron oxide colloidal particles commonly have a black to black-green color (contain Fe3U4) or a reddish- yellow color (contain Fe2U3) in the ochre form.

Metal oxide coated silica microspheres can have a similar color which can be used for identification, whereas cobalt oxide-coated silica microspheres have a blue-purple color.

The indicator or conjugate comprises an analyzing moiety, illustratively here E. Coli 0157 :H7 antibody, "labeled" with the colored metal oxide or metal oxide-coated particles (labeled receptor) . The analyzing moieties can be bound with the metal oxide particles or metal oxide-coated particles by chemical bonding or by physical means such as adsorption, which is preferred. Once labeled, the analyzing moieties are available to react with the analyte, here antigen expressed on the surface of the E. Coli 0157 :H7, and specifically bind same. The colloidal suspension containing colored metal oxide labels bound to analyzing moieties is then dried on the surface of the testing medium.

In a particularly preferred embodiment as illustrated in FIG. 2 and FIG. 3, the lateral flow device 1 comprises five basic components: a plastic backed nitrocellulose (PBNC) membrane 2, a conjugate pad 4, a sample pad 6, a wicking pad 8 and a plastic laminate 10.

The thickness, length and width of the device and its component pad materials directly impact the device design (i.e., sample size), inasmuch as the dimensions of the device dictate the amount of time that taken for the sample to flow through the device. However, such flow characteristics are well known to skilled workers and are available from suppliers of the materials used. In this embodiment, the sample pad is designed to hold about 70 to about 150 μL of aqueous sample. The conjugate pad 4 preferably has a capacity of about 30 to about 75 μL.

An aqueous sample is introduced to the sample pad 6 material prior to the conjugate pad 4. When approximately 100 μL of specimen are added to the sample pad 6, the liquid wets the entire sample pad 6 in three dimensions. Then, the liquid starts to transfer to the conjugate pad 4 communicating across the small overlapping area 12. As the liquid flows across the conjugate pad, it comes in contact with an indicator comprising a metal oxide label grafted to an analyzing moiety, which is disposed on the conjugate pad. In this example, very dark colored colloidal iron oxide particles are used. The colored metal oxide particles act as a colorimetric indicator of the presence of the analyte in the sample .

The labeled analyzing moiety specifically binds a target analyte if present in the sample to form an indicator-analyte complex.

As the conjugate pad 4 becomes saturated, it releases liquid sample to the PBNC membrane 2, thereby carrying the complexes to the PBNC material . Affixed to the PBNC membrane 2 in a predetermined array such as a linear array is a capture moiety 7 and a control moiety 9, which is located parallel to and distal (downstream from) the capture moiety. The wick material 8 in contact with the upper portion of the PBNC membrane serves to draw liquid through the device by capillary action.

As the sample flows through the PBNC membrane 2, the indicator-analyte complex is specifically bound to the capture moiety 7, causing colored metal oxide particles to accumulate there for visual or densitometric evaluation. The presence of the characteristic color of the metal oxide label indicates the presence of the particular analyte to which the assay is directed. The control moiety 9, interacts with some other indicator or with the analyte or to some other moiety or analyte known to be present in the sample or disposed on the device to confirm that the sample has permeated the device. Care is taken not to over saturate and flood the device, which increases the likelihood of false positives, and decreases the accuracy of the assay. Conversely, if too little sample is added, incomplete flow is likely.

An exemplary PBNC membrane 2 is commercially available from Millipore Corporation (Bedford, MA) and sold under the name "hi flow. " An illustrative conjugate pad 4 is a non-woven polyester sold by Ahlstrom Paper Group (Mt . Holly Springs, PA) as product No. 6613. A useful sample pad 6 is Ahlstrom™ Paper Group 609 -- "non woven glass fiber". The wicking pad 8 is Ahlstrom™ Paper Group -- 901 "cotton inter". One plastic laminate 10 is a 0.01 inch vinyl laminate with an adhesive on one side and is sold by G&L Precision Plastics (San Jose, CA) . The plastic laminate holds the other pads and PBNC membrane in place. Other types of sample pad and/or conjugate pad material such as glass fiber, cotton, or synthetic fibers with similar characteristics can also be used in the device. In addition, a simple device can be prepared on an alumina-coated chromatographic sheet as are available from several suppliers .

A. PBNC Membrane Preparation A capture antibody, for example an antibody to E. coli 0157 :H7, and a control antibody, for example goat anti-mouse IgG, are dispensed on the PBNC membrane 2 at a concentration of 0.5 to 2.0 μg/cm in a low molarity buffer, for example 0.005 to 0.2 molar KH2 O4. More particularly, the anti-E. coli 0157 :H7 antibody is dispensed at 1 μg/cm at a concentration of 0.85 mg/mL in 0.015 M KH₂P0₄. The control antibody is dispensed at 1 μg/cm at a concentration of 1.0 mg/mL in 0.1 M phosphate buffered saline.

The control antibody is affixed to the PBNC membrane 2 in a linear array, perpendicular to the direction of flow of the sample. The control antibody is disposed parallel to and distal the capture antibody. The capture antibody is present to confirm that sample is indeed flowing through the device .

Various geometric arrangements or other predetermined arrays, such as for example a circular array, can be employed in conjunction with the characteristic color of the particular metal oxide indicator. A plus/minus configuration can also be used to indicate the presence or absence of the analyte. In such a configuration, a negative sign is depicted in the indicator region and the capture moiety is affixed in a position perpendicularly crossing the minus sign such that if the analyte is present, a plus sign is formed by the accumulation of colored metal oxide indicators at the capture moiety. In a preferred embodiment, iron oxide is used with its characteristic black to black-green color as the indicator. The ochre form of iron oxide, which is reddish-yellow, also can be used, as can any of a variety of other metal oxides, including but not limited to chrome oxide, nickel oxide, aluminum oxide, cobalt oxide, titanium oxide, copper oxide, zinc oxide, europium oxide, and erbium oxide.

After dispensing, the membrane is permitted to dry for approximately 4-24 hours at a typical temperature of about 20 to about 50 °C and at less than about 25% RH. In this example, the membrane is permitted to dry overnight (about 18 hours) at < 25°C and <50% RH.

After drying, the PBNC membrane is blocked to improve the stability of the antibodies on the membrane and to prevent or reduce non-specific binding of the antibody to the membrane. Typically, blocking involves immersing the membrane in one or more solutions designed to achieve these attributes.

In this example, three (3) different solutions are used for blocking. The first is a 0. IM Lysine in 0.015 M KH2PO4, pH 7.4, which is prepared by dissolving 18.3 g of L-lysine monohydrochloride and 2.04 g of KH2PO4 in distilled water and adjusting pH to 7.4. The second is a casein/goat serum solution, which is prepared by dissolving 3 g/L casein in 0.015 M KH2PO4, pH 7.4, and then mixing equal parts of this solution with heat inactivated goat serum that had been filtered through a membrane having pores of 0.2 μm. The third solution is 2% sucrose in 0.015 M KH2PO4, pH 7.4, which is prepared by dissolving 20 g of sucrose in 1 L of 0.015 M KH2PO4 and adjusting pH value to 7.4.

The blocking procedure comprises immersing the membrane in the blocking solutions in a timed sequence, which in this example is as follows:

After blocking, the membrane is permitted to dry overnight (about 18 hours) at < 25°C and <50% RH.

B . Wicking Pad Preparation

The wicking pad is placed over a small portion of the top (control) end of the PBNC membrane when preparing the device lamination. This material is invariably not treated and serves to promote capillary flow through the device. C . Conjugate Pad Preparation

The conjugate pad 4 is placed over a small portion of the control antibody end of the PBNC membrane when preparing device lamination. Prior to dispensing, the conjugate pad material can be blocked to promote release of the conjugate and to protect the antibody (i.e., stability) after drying. Specifically, five (5) different solutions are used for blocking conjugate pad:

Blocking procedure comprises immersing the conjugate pad material in these solutions in a timed sequence as follows:

After blocking, the conjugate pad is permitted to dry overnight (about 18 hours) at <25°C and <50% RH and then blocked by immersion in the following solution for 5 minutes:

After blocking, the conjugate pad 4 again is permitted to dry at least overnight (about 18 hours) at <25°C and <50% RH, after which time the pad 4 is ready for conjugate dispensing.

The conjugate solution is prepared by adding analyzing antibody (KPL, Allentown, PA) to a buffered iron oxide particle colloidal solution at a (final) concentration of 0.01-0.5 mg/mL. These iron oxide particles are commercially available from a variety of sources, including Eichrom Industries, Inc. of Darien, Illinois. The analyzing antibody, which acts as the analyzing moiety, adsorbs on to the surface of the metal oxide particles via borate ions, thereby labeling the particles.

Prior to dispensing on previously blocked and dried conjugate pad 4, this conjugate solution can be modified further to promote both conjugate release and stability after drying.

The conjugate is prepared by first adding 0.05 mg/mL of antibody to the solution containing 0.05 g/mL iron oxide particles in 0.005 M boric acid, pH 9.0. This solution is permitted to mix at least 2 hours and then permitted to stand overnight (about 18 hours) preferably at about 2-8°C prior to the preparation and dispensing of the final conjugate solution

The final conjugate solution is prepared as follows :

The conjugate solution is dispensed on the previously blocked conjugate pad 4 at a concentration of 0.01 to 0.05 mL/cm. Preferably, the final conjugate solution is dispensed on the blocked conjugate pad 4 at a concentration of approximately 0.029 mL/cm and permitted to dry at least overnight (about 18 hours) at <25°C and <50% RH. The conjugate pad 4 is then ready for use in the device.

D. Sample Pad Preparation

The sample pad 6 is placed over all or a portion of the conjugate pad when preparing the device lamination. This pad 6 serves to meter the sample flow. It can be used in untreated or "virgin" condition or after blocking depending on the specific materials properties.

Two different solutions are used for blocking sample pad material :

1. Casein/Goat Solution (prepared as previously outlined for PBNC membrane blocking)

2. 0.1 M Lysine/0 015 M KH₂P0₄ Solution

(prepared as previously outli .ned for PBNC membrane blocking) , then admixed with 4 g/L sucrose, 9.2 g/

NaCl and 50 mL gelatin.

Blocking procedure comprises immersing the sample pad material in the Casein/Goat solution for 5 minutes followed by immersion in the Lysine/0.015 M

KH2PO4 solution for 5 minutes. After blocking, the sample pad 6 is permitted to dry at least overnight (about 18 hours) at <25°C and <50% RH and is then ready for use in the device .

II . Comparative Sensitivity vs. Colloidal Gold As shown in FIGS. 4 and 5, in cross- reactive comparisons, an iron oxide colorimetric indicator in accordance with the present invention has been found to be superior to colloidal gold at discriminating between E. coli 0157 :H7 as compared with E. coli 0157 :H38 and E. coli H45 in side by side testing done at room temperature.

At a concentration of 10^ colony- forming units per milliliter ( "CFU' s/mL" ) , the relative intensity of the indicator for E. coli 0157 :H7 was approximately twice the intensity of E. coli 0157 :H38 and H45 respectively, at a relative intensity ratio of approximately 4:2:2. In contrast, when colloidal gold was used as the indicator, there was very little difference between relative intensities for E. coli 0157 :H7 as compared with H38 and H45 at a ratio of approximately 4:4:4.

At a concentration of 10⁷ CFU's/mL, the intensity differential for the iron oxide indicator was even more pronounced with the relative intensity of the E. coli 0157 :H7 nearly three times that of H38 at a ratio of approximately 3:1 and over three times that of H45 at a ratio of approximately 3:0.1. Colloidal gold again showed almost no difference between the relative intensities of E. coli 0157 :H7, 0157:H38 and 0157:H45 at a ratio of 4:4:4. At a concentration of 10⁶ CFU's/mL, the iron oxide indicator was measured at a relative intensity of 1 for E. coli 0157 :H7. The intensities for E. coli 0157 :H38 and H:45 were negligible. At this concentration, colloidal gold was measured at a relative intensity of 3 for E. coli 0157 :H7 as compared with relative intensities of 2.5 for E. coli 0157 :H38 and H45 commercially available from Neogen, East Lansing, MI

III . Comparative study v. commercially available lateral flow devices Current lateral flow or rapid immunochromatographic color change tests on the market for detecting the pathogen E. coli 0157 :H7 are available in two basic forms: polyclonal (antibody) based or monoclonal based. These test generally use colored colloidal gold-antibody conjugation as the means of visual detection. In evaluating the efficacy of these types of assays and considering the virulence of E. coli 0157 :H7 a discussion of the issue of sensitivity and specificity for detecting this pathogen is in order.

Sensitivity is for the most part related to the number of, and accessibility to, specific epitopes (antibody specific binding sites) present on the surface of the pathogen or cell being targeted (i.e. the analyte) . The colored conjugate binds to these sites which ultimately give rise to visual detection if enough cells are present. It can be reasonably argued that the monoclonal system, which is designed to target a single epitope, might exhibit lower sensitivity due to low epitope abundance. This would lower the total amount of colored conjugate- cell binding limiting the color accumulation, thus lowering the overall sensitivity. There are also kinetic issues associated with efficient conjugate- cell contact when low abundance surface epitopes are targeted, further limiting the ability of the colored conjugate to even "see" the epitope during the course of the test. Polyclonal systems by definition target multiple epitopes and would not suffer from these issues nearly to the same degree.

Specificity, however, is solely dependent on an antibody targeting specific epitopes present only on the target cell of interest and on no other related species. Antibodies by nature can also bind to particles to form conjugates in such a way that there is an orientation or directionality to the active site on the antibody. Antibodies are known by their characteristic "Y" configuration where the "V" portion is known as the F(_ab-)₂ portion, and the "I" base portion known as the F_c portion. It is each of the individual F(_ab) fractions [paratope-containing antibody portions "\" and "/"] of the F_(ab-₎₂ ["V"] portion that imparts to the antibody its high immunospecific properties.

Because antibodies are glycoproteins, binding to particles can occur in the following basic ways :

(1) Inverted or F_(ab-)₂ portion down "Λ" orientation;

(2) Sideways or horizontal "<" orientation where the F_c portion and one or more of the F<_ab) fractions is attached; or,

(3) Vertical or F_c bound "Y" orientation. The latter orientation (3) is the preferred one as this optimally orients the highly immunospecific portion of the antibody for maximum interaction with the desired surface epitope (s) to be detected. Along with other hydrophobic interactions that may be present, it is believed that the preponderance of (1) and (2) in the conjugate formation causes increased non-specific adsorption and reduced immunospecificity in these types of systems. In addition, because the hydrophilic sugar (glyco) moiety is located on the F_c portion, optimum orientation is promoted with highly hydrophilic surfaces. It is possible to increase sensitivity at the expense of specificity by not accounting for proper orientation of the antibody.

For immunochromatographic assays, low sensitivity for a target cell is thought to lead to an increase in false negative results, and high(er) sensitivity for non-target cells (i.e., lower specificity) is thought to lead to an increase of false positives.

A basic calculation assuming a level of contamination of only one viable E. coli 0157 :H7 colony forming unit (CFU) in a sample that is then cultured in 225 mL of medium to enrich the population shows that 28 cell doublings are required to just exceed a total bacterial count of 10⁶ CFU's/mL. Using standard commercially available media having a typical lag time of 5 hours prior to the onset of cell division and a CFU doubling time of 30 minutes thereafter would then require a total of 19 hours to reach this 10⁶ CFU's/mL contamination level. Moreover, if (commercially available) optimized medium is used that typically reduces the lag time to about one hour and increases the double rate by a factor of two, this 10⁶ CFU's/mL bacterial count is reached in only 8 hours .

What appears evident from this analysis is that, if a sample is contaminated at any level, using either fast growth medium or conventional 20 hour medium, a 10^s CFU's/mL sensitivity level should be more than sufficient to observe a positive test. If a test does not have a minimum 10⁶ CFU's/mL sensitivity it, by definition, will miss contamination levels of less than about 5 CFU' s per sample, regardless of how specific the test is and which culture media is used.

It is believed that a suitable assay needs only a 10⁶ CFU's/mL sensitivity level to ostensibly eliminate false negatives using either the accelerated or standard growth medias available. Nothing is believed to be inherently gained by having higher sensitivity unless the medium is not performing correctly or the culturing was done improperly. One viable E. coli 0157 :H7 cell per sample will produce at least 10⁶ CFU's/mL using standard culturing protocols. With lateral flow devices, false negatives, therefore, should not be a real issue if the test meets the minimum sensitivity guidelines .

With respect to false positives, it is apparent that the test must be exhibiting a high degree of non-specific binding and thus is highly sensitive for non :H7 species. The more non-specific binding, the more likely will be the incidence of a false positive.

With the variety of currently available markers and the increasing number of available antibodies, it is believed that the specificity of the test should be maximized for optimum performance, reliability and acceptability of the test for point- of-use clinical as well as non-clinical applications. High sensitivity is an important consideration, but reduction of false positives (specificity) will be one of the primary hurdles to general acceptance of the test.

Lateral flow devices generally require only a sensitivity level of about 10⁶ CFU's/mL to be highly effective for detecting samples contaminated with the E. coli 0157 :H7 pathogen. Less sensitivity can cause an increase in false negatives by missing contamination levels of less than about 5 CFU' s per sample. Maximizing specificity and not maintaining the 10⁶ CFU's/mL sensitivity generally is not desirable as the false negative issue far outweighs the false positive issue. Lower specificity at the appropriate minimum detection level will increase the number of false positives and the inherent costs in this can be substantial. The issue then is less an issue of balance between sensitivity and specificity of the test, but ensuring the test has maximum specificity at approximately the 10⁶ CFU's/mL or 1 cell per sample contamination level with the highest specificity possible.

Testing Results Comparative testing was performed on three prominent suppliers of E. coli 0157 :H7 lateral flow rapid color change tests on the market. To evaluate sensitivity, tests were performed on 4 different E. coli 0157 :H7 strains tested down to levels of 10⁵ CFU/mL (CFU = colony forming unit) . To evaluate specificity, several E. coli O157:non-H7 species were evaluated. It was shown that there exists a strong inverse correlation between sensitivity and specificity for these devices. The most sensitive test exhibits the least specificity and vice versa.

Sensitivity Results. One manufacturer [MFR "A"] using a colloidal gold polyclonal antibody based rapid test, shows detection limits (sensitivity) for 4 different E. coli 0157 :H7 strains down to levels of approximately 10⁵ CFU/mL. These data were collected starting with a 10⁹ CFU/mL EC culture broth followed by four 10-fold serial dilutions with PBS. A second manufacturer [MFR "B"] also a polyclonal rapid test but using latex particles, shows detection limits for the same four strains of 10⁵'⁵ CFU/mL; roughly a 10 fold lower sensitivity than MFR "A" and substantially similar to the test based using the inventive metal oxide indicators.

FIG. 6 illustrates the sensitivity of the metal oxide indicator test for E. Coli 0157 strains at concentrations of 10⁶ CFU/mL (the first bar of each pair of bars per strain, viewing from left to right) and 10⁵"⁵ CFU/mL (the second bar of each pair of bars) . The E. Coli 0157 strains from obtained from the University of Pennsylvania E. Coli Reference Laboratory. A third manufacturer [MFR "C" ] provides a colloidal gold-linked monoclonal antibody-based rapid test that produces a strong positive test at 10⁵"⁵ CFU/mL for only one of the four strains and very weak positives or negative results for the other three; a 10-100-fold decrease in sensitivity compared to "A". What appears to arise from these data is that the polyclonal systems have noticeably higher sensitivity than the monoclonal system. Specificity Results. Because all three manufacturers, as well as the contemplated metal oxide indicator test described herein, exhibit strong positives for the :H7 strain at 10⁶ CFU/mL, the specificity for each test can then be compared by looking at the detection of E. coli 0157 :non-H7 species at the same 10⁶ CFU/mL concentration. Figure 7 illustrates this showing that the most sensitive polyclonal test for the :H7 species ["A"] (the second bar of each tripartite group of bars per strain, viewing from left to right) , readily detects all 10 :non-H7 strains at 10⁶ CFU/mL, some more weakly than others. The other polyclonal system ["B"] (the third bar of each tripartite group of bars per strain) detects all 10 as well. As can be seen from FIG. 7, a contemplated metal oxide indicator assay (the first bar of each tripartite group of bars per strain) exhibits lower sensitivity for the "non :H7" species (H:29, H:32 and H:44) compared to the other similar polyclonal based tests ["A" & "B"] and is even negative for 3 of the strains. This clearly indicates the improved specificity of the metal oxide indicators over either colloidal gold or latex indicators using polyclonal antibodies .

Examining the monoclonal system ["C"] for detection of :non-H7 species surprisingly shows that this system, while designed to be highly specific for E. coli 0157 :H7, exhibits significant sensitivity for several of the :non-H7 strains [FIG. 8] . Moreover, when compared to the specificity of the metal oxide indicator polyclonal test, the monoclonal based test exhibits even higher sensitivity for two of the :non- H7 strains. In one case [:H32], "C" (the second bar of each pair of bars per strain, viewing from left to right) readily detects this strain whereas the metal oxide indicator polyclonal test (the first bar of each pair of bars per strain) does not. These results are readily seen from examination of FIG. 8.

For polyclonal systems, there is inherently a less overall specificity expected by virtue of the presence of multiple immunospecific antibodies for different surface antigen sites. The ability to detect other strains of E. coli 0157 would not be surprising. What is surprising is that the monoclonal system exhibits as much nonspecific adsorption as it does. There are many factors that can influence the specificity of a label-antibody conjugate detection system for lateral flow devices. The sensitivity and specificity data shown above comparing polyclonal and monoclonal conjugates, using either gold or latex particles, clearly illustrates that there is more to specificity than just the nature of the antibody used. The label choice has a strong influence in the specificity of the test as well. It is evident from the data above that the choice of the label to improve antibody orientation can dramatically increase specificity of a polyclonal based system. The primary emphasis on increased specificity in a test lies in the notion that one only needs 10⁶ CFU/mL detection limits for the E. coli 0157 :H7 bacteria in routine testing. Proper culturing can achieve this concentration level in almost any sample. What is needed in a test is the higher specificity while still maintaining a suitable lower limit of detection, to significantly reduce the chances for a false positive result. In essence, for the existing tests on the market, the sensitivity- specificity balance is really a balance between false negative-false positive test results that one is willing to accept for detecting E. coli 0157 :H7. Proper culturing essentially takes care of the sensitivity issue; thus, optimizing a test for specificity is the key. As demonstrated in FIGs 6-8, a contemplated metal oxide indicator test was found to provide the highest specificity at the desired sensitivity for the standard detection of E. coli 0157 :H7 as compared with the three commercial tests that were studied.

IV. Additional Alternative Embodiments

Although it is preferred to employ the five components in a lateral flow device as described above the inventive device can employ fewer or greater numbers of layers as well. For example, where only four layers are used, the separate sample pad is eliminated and longer conjugate and wicking pads are used. In a six or more layer device, additional sample pads can be added, which serve to reduce the flow rate of the sample through the device and permit for more prolonged exposure of the analyte to the analyzing moiety. A single layer device also is contemplated, with the analyzing moiety at one end and the capture moiety at the other end.

Those skilled in the art will readily recognize that varying combinations of layers, pads, moieties, analytes and controls can be employed without deviating from the spirit of the invention, with the analyzing and capture moieties generally comprising specific binding partners for the analyte. As noted above, the moieties and analytes can comprise a variety of ligands including antibodies, antigens, DNA, RNA. Thus, for example, if the device is used as a pregnancy assay, the analyte can be human chorionic gonadotrophin, which would be present in a pregnant woman's urine sample, and the analyzing moiety an antibody to human chorionic gonadotrophin such as, for example, KEP-1E5.1 (ATCC HB 8095, American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852-1776) and a capture antibody that is another specific antibody to human chorionic gonadotrophin.

In another example, the device is used in an assay for the Epstein-Barr virus with a human blood serum sample. In such an example, goat- antihuman antibody disposed iron oxide, is the analyzing moiety for Epstein-Barr Nuclear Antigen

(EBNA) . The capture moiety is polypeptide P62 antigen array. A general description of an EBNA assay employing these antibody and antigen combinations is contained in U.S. Patent No.

5,122,448 (Vaughan et al . ) , the disclosure of which is incorporated by reference.

In another example, nuclear matrix proteins (NMPs) are employed as analyzing moieties for malignant cells, as for example is disclosed in U.S.

Patents No. 5,866,535 (Getzenberg et al . ) , No.

5,273,877 (Fey et al . ) and No. 4,882,268 (Penman et al . ) , each of which is incorporated by reference. As noted above, the device can be used in conjunction with a variety of antigen-antibody pairs, which are commercially available from a number of sources, including: Biospacific; Dako; Pharmigen;

Serotec; Biodesign; Oncogene Research Products; Zymed; Alexis Biochemicals; and KPL, which supplies anti-E. coli 0157 :H7 antibodies.

A control moiety, if present, can also be a specific binding partner for the analyte, or alternatively can be some other sort of reactive substance that indicates that the sample has permeated the test device (e.g. pH indicator).

In an alternative embodiment, no control antibody is used. Instead, identical or different antibodies are used for analyzing and for capture, with the analyzing antibody selectively reactive to a first site on an analyte antigen and the capture antibody selectively reactive to a second site on that antigen.

Rather than using an aqueous sample, it is contemplated that a non-liquid or anhydrous sample can alternatively be used. Water or other exogenous transporting liquid then can be added to the sample after it has been contacted with the lateral flow device to transport the sample via capillary action from the first portion to the capture moiety disposed on the second portion.

The capture moiety can be visible or invisible to the naked eye. However, if visible, it is preferred that the capture moiety be of a color and intensity such that the presence of a colorimetric indicator still can be discerned visually. The assay also can be quantitatively analyzed, such as a densitometer test, to determine the amount or concentration of analyte present by the density of the indicator present.

Each of the patents and articles cited herein is incorporated by reference. The use of the article "a" or "an" is intended to include one or more .

Those skilled in the art will recognize that the invention claimed herein, which is defined by the scope of the claims set forth below, is not limited to the above disclosed embodiments. Thus, it will be readily apparent to those skilled in the art that additional modifications and alternative embodiments may be developed without departing from the teachings and spirit of the claimed invention.

Claims

What is claimed is

1. An assay indicator comprising a metal oxide label grafted to an analyzing moiety that specifically binds an analyte when present in a sample, said indicator signaling the presence of the analyte in the sample.

2. The indicator of claim 1 wherein said label is comprised of a metal oxide microparticle .

3. The indicator of claim 2 wherein said metal oxide is iron oxide.

4. The indicator of claim 1 wherein said label is comprised of a metal oxide coated, non-porous silica microparticle.

5. The indicator of claim 4 wherein said metal oxide is iron oxide.

6. The indicator of claim 1 wherein said indicator signal is provided by a colored metal oxide label.

7. The indicator of claim 1 wherein said label is selected from the group consisting of iron oxide, copper oxide, titanium oxide, cobalt oxide, nickel oxide and chromium oxide .

8. The indicator of claim 1 wherein said label is magnet responsive.

9. A lateral flow device comprising: an assay medium having a first portion and a second portion in flow communication with each other; an indicator for identifying the presence of an analyte in a sample comprising a metal oxide label grafted to an analyzing moiety that specifically binds said analyte, said indicator initially disposed on said first portion of said assay medium; a capture moiety that specifically binds said analyte affixed to said second portion of said assay medium, said indicator flowing to and accumulating at said capture moiety to signal the presence of said analyte when a sample containing said analyte is introduced to said first portion of said assay medium.

10. The lateral flow device of claim 9 wherein said analyzing moiety is an antibody and said analyte is an antigen.

11. The lateral flow device of claim 9 wherein said capture moiety is an antibody that specifically binds said analyte.

12. The lateral flow device of claim 9 wherein said metal oxide label is selected from the group consisting of iron oxide, copper oxide, titanium oxide, cobalt oxide, nickel oxide and chromium oxide.

13. The lateral flow device of claim 9 wherein said capture moiety is affixed to said second portion of said assay medium in a predetermined array.

14. The lateral flow device of claim 9 further comprising a third portion in flow communication with said first portion, said third portion receiving said aqueous sample containing said analyte and communicating said analyte to said first portion.

15. The lateral flow device of claim 14 further comprising a wicking portion in flow communication with said second portion for promoting capillary flow of said sample through the device.

16. The lateral flow device of claim 15 wherein said first, second, third and wicking portions are generally planar.

17. The lateral flow device of claim 14 wherein said third portion is blocked to limit non-specific binding between said sample and said third portion.

18. The lateral flow device of claim 9 wherein said analyzing moiety and said capture moiety comprise an antibody to E. coli 0157 :H7, and said analyte is E. coli 0157 :H7.

19. The lateral flow device of claim 9 wherein said analyzing moiety and said capture moiety comprise antibodies to human chorionic gonadotrophin, and said analyte is human chorionic gonadotrophin .

20. The lateral flow device of claim 9 wherein said first and second portions are blocked to limit non-specific binding between said sample and said portions and to facilitate the flow of said indicator across said portions.

21. The lateral flow device of claim 9 further comprising a control moiety disposed on said second portion.

22. The lateral flow device of claim 9 wherein said indicator signal is provided by a colored metal oxide label .

23. The lateral flow device of claim 9 wherein said indicator signal is provided by a radioactive metal oxide label .

24. The lateral flow device of claim 9 wherein said indicator signal is provided by a magnet responsive metal oxide label.

25. The lateral flow device of claim 9 wherein said indicator signal is provided by a fluorescent metal oxide label .

26. A lateral flow device comprising a multilayer assay medium having a first layer and a second layer in flow communication with each other, said first layer having an analyzing antibody bound to a colorific indicator comprising a plurality of colored colloidal metal oxide particles, said analyzing antibody specifically binding an antigen when an aqueous sample containing said antigen is introduced to said first portion of said assay medium; said second layer in overlapping flow contact with a portion of said first layer, said second layer having a capture antibody affixed to a first portion thereof and a control antibody affixed to a second portion thereof, said capture and control antibodies affixed to said second layer specifically binding said antigen when said sample containing said antigen contacts said affixed antibodies.

27. The lateral flow device of claim 26 further comprising a third layer in flow communication with said first layer for receiving said aqueous sample containing said antigen and communicating same to said first pad.

28. The lateral flow device of claim 27 further comprising a wicking layer for promoting capillary flow of said sample through the device, said wicking layer in flow communication with said second layer and located distal from the contacting portions of said first and second layers.

29. A lateral flow assay method comprising the steps of: a) introducing an aqueous sample containing an analyte to the lateral flow device of claim 9 to form indicator-analyte complexes when said analyte is present in said sample; b) delivering via capillary action said complexes to a capture moiety disposed in a predetermined array on said second portion of said assay medium; c) capturing a population of indicators with attached complexes at said capture moiety to signal the presence of said analyte based on a characteristic property of said metal oxide label.

30. The lateral flow assay method of claim 29, wherein said capture moiety is arranged in a linear array.

31. The lateral flow assay method of claim 30 wherein said characteristic property is color.

32. A lateral flow assay method comprising the steps of : a) contacting the lateral flow device of claim 9 with an anhydrous sample; b) adding liquid to said sample; c) forming indicator-analyte complexes, when said analyte is present in said sample; d) employing said liquid to deliver via capillary action said complexes to a capture moiety disposed in a predetermined array on said second portion of said assay medium; e) capturing a population of indicators with attached complexes at said capture moiety to signal the presence of said analyte based on a characteristic property of said metal oxide label .

33. A lateral flow device comprising: an assay medium having a first portion and a second portion in flow communication with each other; a colorific indicator comprising a plurality of metal oxide particles grafted to antibodies that are selectively reactive with an antigen, said indicator initially disposed on said first portion of said assay medium; a plurality of capture antibodies that are selectively reactive with said antigen affixed to said second portion of said assay medium, said indicator flowing to and accumulating at said capture antibody to signal the presence of said antigen when an aqueous sample containing said antigen is introduced to said first portion of said assay medium.

34. The lateral flow device of claim 33 wherein said metal oxide particles are iron oxide particles.

35. The lateral flow device of claim 34 wherein said iron oxide particles are magnet responsive.