WO2001018235A2 - Dispositif a flux lateral a indicateur a oxyde metallique et procede de bioanalyse employant ce dispositif - Google Patents

Dispositif a flux lateral a indicateur a oxyde metallique et procede de bioanalyse employant ce dispositif Download PDF

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Publication number
WO2001018235A2
WO2001018235A2 PCT/US2000/040859 US0040859W WO0118235A2 WO 2001018235 A2 WO2001018235 A2 WO 2001018235A2 US 0040859 W US0040859 W US 0040859W WO 0118235 A2 WO0118235 A2 WO 0118235A2
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Prior art keywords
indicator
lateral flow
analyte
metal oxide
flow device
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PCT/US2000/040859
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English (en)
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WO2001018235A3 (fr
WO2001018235A8 (fr
Inventor
L. Allen Ross
Raymond L. Miller
Timothy J. Barder
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Eichrom Technologies, Inc.
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Priority to AU12510/01A priority Critical patent/AU1251001A/en
Publication of WO2001018235A2 publication Critical patent/WO2001018235A2/fr
Publication of WO2001018235A8 publication Critical patent/WO2001018235A8/fr
Publication of WO2001018235A3 publication Critical patent/WO2001018235A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/10—Magnetic particle immunoreagent carriers the magnetic material being used to coat a pre-existing polymer particle but not being present in the particle core

Definitions

  • 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.
  • 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” .
  • a single 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.
  • 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.
  • antibody 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.
  • glycoproteins glycosylated proteins
  • 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.
  • 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.
  • 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 .
  • the analyzing and capture moieties are the same or different.
  • 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.
  • 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 123 I, 1 5 I, 126 I, 128 I, 131 I, 13 1, and 51 Cr. Positron emitting elements such as X1 F, 18 F, 15 0 and 13 N 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.
  • 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.
  • HCG human chorionic gonadotrophin
  • the antibodies 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • metal oxide indicators are hydrophilicity.
  • 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.
  • 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.
  • 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.
  • 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
  • FIG. 8 is a chart comparing a contemplated Metal Oxide Indicator E. coli 0157 :H7 Test with commercial monoclonal test.
  • 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 ⁇ m to about 10 ⁇ m.
  • 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.
  • metal oxide particles in a colloidal suspension, are introduced to any of a variety of analyzing moieties (receptors) .
  • a suspension is often referred to as a "ferro-fluid" where the particles are iron oxide.
  • 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.
  • particles, preferably silica microspheres having a diameter of about 0.1 ⁇ m to about 10 ⁇ m, coated with a metal oxide can be used as indicators .
  • 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.
  • 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.
  • PBNC plastic backed nitrocellulose
  • 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.
  • the sample pad is designed to hold about 70 to about 150 ⁇ L of aqueous sample.
  • the conjugate pad 4 preferably has a capacity of about 30 to about 75 ⁇ L.
  • aqueous sample is introduced to the sample pad 6 material prior to the conjugate pad 4.
  • 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.
  • an indicator comprising a metal oxide label grafted to an analyzing moiety, which is disposed on the conjugate pad.
  • 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.
  • the conjugate pad 4 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.
  • 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 AhlstromTM Paper Group 609 -- "non woven glass fiber”.
  • the wicking pad 8 is AhlstromTM 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.
  • 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.
  • a simple device can be prepared on an alumina-coated chromatographic sheet as are available from several suppliers .
  • a capture antibody for example an antibody to E. coli 0157 :H7
  • a control antibody for example goat anti-mouse IgG
  • the anti-E. coli 0157 :H7 antibody is dispensed at 1 ⁇ g/cm at a concentration of 0.85 mg/mL in 0.015 M KH 2 P0 4
  • the control antibody is dispensed at 1 ⁇ g/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 .
  • a plus/minus configuration can also be used to indicate the presence or absence of the analyte.
  • 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.
  • 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.
  • the membrane 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.
  • the PBNC membrane 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.
  • blocking involves immersing the membrane in one or more solutions designed to achieve these attributes.
  • 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 ⁇ m.
  • 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:
  • the membrane After blocking, the membrane is permitted to dry overnight (about 18 hours) at ⁇ 25°C and ⁇ 50% RH.
  • 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.
  • 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:
  • 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:
  • 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.
  • analyzing antibody KPL, Allentown, PA
  • 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.
  • this conjugate solution 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.
  • 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.
  • 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.
  • 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
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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( a b-) 2 portion, and the "I” base portion known as the F c portion. It is each of the individual F( a b) fractions [paratope-containing antibody portions " ⁇ " and "/"] of the F (ab - )2 ["V”] portion that imparts to the antibody its high immunospecific properties.
  • orientation (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.
  • the preponderance of (1) and (2) in the conjugate formation causes increased non-specific adsorption and reduced immunospecificity in these types of systems.
  • 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.
  • a suitable assay needs only a 10 6 CFU's/mL sensitivity level to ostensibly eliminate false negatives using either the accelerated or standard growth medias available. None 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 10 6 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 .
  • Lateral flow devices generally require only a sensitivity level of about 10 6 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 10 6 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 10 6 CFU's/mL or 1 cell per sample contamination level with the highest specificity possible.
  • 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 10 5 CFU/mL. These data were collected starting with a 10 9 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 10 5 ' 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 10 6 CFU/mL (the first bar of each pair of bars per strain, viewing from left to right) and 10 5 " 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 10 5 " 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".
  • 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 10 6 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.
  • 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 .
  • 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.
  • the analyzing and capture moieties generally comprising specific binding partners for the analyte.
  • the moieties and analytes can comprise a variety of ligands including antibodies, antigens, DNA, RNA.
  • 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.
  • KEP-1E5.1 ATCC HB 8095, American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852-1776
  • a capture antibody that is another specific antibody to human chorionic gonadotrophin.
  • the device is used in an assay for the Epstein-Barr virus with a human blood serum sample.
  • goat- antihuman antibody disposed iron oxide is the analyzing moiety for Epstein-Barr Nuclear Antigen
  • EBNA 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.
  • NMPs nuclear matrix proteins
  • Patents No. 5,866,535 (Getzenberg et al . ) , No.
  • 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;
  • 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).
  • 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.
  • 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.

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Abstract

L'invention concerne un dispositif à flux latéral pourvue d'un indicateur comprenant une étiquette oxyde métallique greffée à un groupe caractéristique d'analyse biomédicale et un procédé bioanalytique employant ce dispositif. Cette invention emploie une pluralité de particules d'oxyde métallique ou de particules de silice non-poreux revêtues d'oxyde métallique comme étiquettes greffées à des groupes caractéristiques d'analyse qui lient sélectivement l'analyte, et identifient l'analyte auquel la bioanalyse est dirigée. Dans une bioanalyse envisagée, après qu'un échantillon est introduit, le groupe caractéristique d'analyse lie sélectivement l'analyte, si présent, pour former un complexe indicateur d'analyte. Le complexe circule à travers le dispositif par action capillaire vers le région récepteur où un deuxième groupe caractéristique emprisonné apposé au moyen bioanalytique lie le complexe faisant ainsi s'accumuler les oxydes métalliques. Les bioanalyses peuvent alors être interprétées à l'aide d'une caractéristique de propriété d'une étiquette d'oxide métallique telle que la couleur dans la région de réception.
PCT/US2000/040859 1999-09-09 2000-09-08 Dispositif a flux lateral a indicateur a oxyde metallique et procede de bioanalyse employant ce dispositif WO2001018235A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU12510/01A AU1251001A (en) 1999-09-09 2000-09-08 Lateral flow device with metal oxide indicator and assay method employing same

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US15297399P 1999-09-09 1999-09-09
US60/152,973 1999-09-09
US65723300A 2000-09-07 2000-09-07
US09/657,233 2000-09-07

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WO2009126735A1 (fr) * 2008-04-09 2009-10-15 Becton Dickinson And Company Immuno-essais sensibles utilisant des nanoparticules revêtues
CN108267575A (zh) * 2018-01-23 2018-07-10 闽江学院 CuO标记技术用于厚度可控蛋白质分子印迹膜的制备

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US5445970A (en) * 1992-03-20 1995-08-29 Abbott Laboratories Magnetically assisted binding assays using magnetically labeled binding members
US5736188A (en) * 1994-08-08 1998-04-07 Alcock; Susan Printed fluid transport devices
US5998224A (en) * 1997-05-16 1999-12-07 Abbott Laboratories Magnetically assisted binding assays utilizing a magnetically responsive reagent
US6136610A (en) * 1998-11-23 2000-10-24 Praxsys Biosystems, Inc. Method and apparatus for performing a lateral flow assay
US6140136A (en) * 1998-09-18 2000-10-31 Syntron Bioresearch, Inc. Analytical test device and method of use

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Publication number Priority date Publication date Assignee Title
US5445970A (en) * 1992-03-20 1995-08-29 Abbott Laboratories Magnetically assisted binding assays using magnetically labeled binding members
US5424193A (en) * 1993-02-25 1995-06-13 Quidel Corporation Assays employing dyed microorganism labels
US5736188A (en) * 1994-08-08 1998-04-07 Alcock; Susan Printed fluid transport devices
US5998224A (en) * 1997-05-16 1999-12-07 Abbott Laboratories Magnetically assisted binding assays utilizing a magnetically responsive reagent
US6140136A (en) * 1998-09-18 2000-10-31 Syntron Bioresearch, Inc. Analytical test device and method of use
US6136610A (en) * 1998-11-23 2000-10-24 Praxsys Biosystems, Inc. Method and apparatus for performing a lateral flow assay

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009126735A1 (fr) * 2008-04-09 2009-10-15 Becton Dickinson And Company Immuno-essais sensibles utilisant des nanoparticules revêtues
CN102016585A (zh) * 2008-04-09 2011-04-13 贝克顿·迪金森公司 使用包被的纳米颗粒的灵敏的免疫测定
JP2011516889A (ja) * 2008-04-09 2011-05-26 ベクトン・ディキンソン・アンド・カンパニー 被覆ナノ粒子を使用した高感度免疫学的検定
JP2015148629A (ja) * 2008-04-09 2015-08-20 ベクトン・ディキンソン・アンド・カンパニーＢｅｃｔｏｎ， Ｄｉｃｋｉｎｓｏｎ Ａｎｄ Ｃｏｍｐａｎｙ 被覆ナノ粒子を使用した高感度免疫学的検定
EP2952897A1 (fr) * 2008-04-09 2015-12-09 Becton, Dickinson and Company Immunoessais sensibles à l'aide de nanoparticules revêtues
CN107561270A (zh) * 2008-04-09 2018-01-09 贝克顿·迪金森公司 使用包被的纳米颗粒的灵敏的免疫测定
CN108267575A (zh) * 2018-01-23 2018-07-10 闽江学院 CuO标记技术用于厚度可控蛋白质分子印迹膜的制备

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