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WO2003052379A2 - Dosage immunologique rapide du rsv - Google Patents

Dosage immunologique rapide du rsv Download PDF

Info

Publication number
WO2003052379A2
WO2003052379A2 PCT/US2002/039999 US0239999W WO03052379A2 WO 2003052379 A2 WO2003052379 A2 WO 2003052379A2 US 0239999 W US0239999 W US 0239999W WO 03052379 A2 WO03052379 A2 WO 03052379A2
Authority
WO
WIPO (PCT)
Prior art keywords
sample
wicking member
antibody
wicking
conjugate
Prior art date
Application number
PCT/US2002/039999
Other languages
English (en)
Other versions
WO2003052379A3 (fr
Inventor
John H. Slack
Charles Radliff
Original Assignee
Integrated Biotechnology Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Integrated Biotechnology Corporation filed Critical Integrated Biotechnology Corporation
Priority to AU2002361677A priority Critical patent/AU2002361677A1/en
Publication of WO2003052379A2 publication Critical patent/WO2003052379A2/fr
Publication of WO2003052379A3 publication Critical patent/WO2003052379A3/fr

Links

Classifications

    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
    • G01N2333/135—Respiratory syncytial virus

Definitions

  • This invention relates generally to immunological testing and, more specifically, to icking immunoassays involving migration of both antigen and antibody.
  • Immunoassays have been known for some time. They make use of the ability of antibodies to bind selectively to specific antigens. Such binding is used in assays in conjunction with a signal technology that enables an antibody-antigen binding event to be identified.
  • One useful signal technology involves the conjugation of small colored particles, such as colloidal gold spheres or plastic beads, with antibodies. Observation of the colored particles can be used to identify the location of the conjugate and to infer binding of the conjugated antibody with an antigen.
  • An immunoassay commonly known as a "sandwich” assay involves such conjugates.
  • sandwich immunoassay a mixture of conjugated antibody/signal moieties and liquid sample solution migrate in a wicking member, such as a paper strip. Antigen in the sample will be bound by the conjugate during migration along the wicking member. The resulting conjugate-antigen moieties are captured by antibodies immobilized at a downstream location on the wicking member, forming a "sandwich” structure in which the antigen is sandwiched between the immobilized antibody and the conjugate. Individual colored particles are normally too small to be visible to the naked eye; however, they become readily visible when concentrated at, for example, the capture location in a sandwich assay.
  • Sandwich immunoassays have become accepted in the medical diagnostics field. Over-the-counter pregnancy tests based on sandwich immunoassays are well known. Sandwich immunoassays involving wicking flow in a matrix, such as filter paper, can provide quick, easy-to-read tests for a variety of antigens.
  • One problem is the criticality of reading some sandwich immunoassays within a relatively short time period in order to obtain accurate results. For example, some immunoassays that accurately show no signal during a critical read period will gradually accumulate unbound conjugate at the capture antibody location, resulting in a false positive reading if the test results ⁇ are examined after the critical read period. In other assays an optical signal will become weaker and less precise over time, resulting sometimes in only a blur of weak color around the location of a signal that was originally unequivocal.
  • a related problem is that the assay device providing such transient results cannot itself serve as a permanent reference of assay results in such assays. It would be desirable to be able to place a test strip in a patient's file for future reference. However; this often has not been possible due to the tendency the signal in strip assays to change or to drift over time.
  • RSV respiratory syncytial virus
  • a lateral flow sandwich immunoassay that, in one aspect, comprises a wicking member contained in a case having a sample- receiving port for applying sample to the wicking member.
  • the sample receiving port is shaped to receive a predetermined sample amount in a configuration that provides a head pressure for moving the sample into the wicking member.
  • the case includes vents to facilitate rapid drying of the sample on the member after the assay is performed and also includes internal structures to prevent constriction of flow in the member by contact with the case.
  • the invention is a device for performing a fast, simple assay for human RSV.
  • the device comprises a wicking member that defines a pathway for sample flow and that releasably holds at a first location an antibody against a first epitope of a fusion protein produced by RSV-infected cells, the antibody being conjugated with a signal particle.
  • a capture antibody for a second epitope of the fusion protein is immobilized at a second location on the wicking member.
  • the wicking member is held in a case that includes a loading port shaped to hold a predetermined amount of sample in a configuration that causes the sample itself to provide head pressure for encouraging movement of the sample into the wicking member.
  • the case also includes vents in the vicinity of the sink area of the wicking member to facilitate drying.
  • the wicking member includes a sample application pad in wicking contact with a conjugate pad that is in turn in wicking contact with a test-reading membrane.
  • the test-reading membrane is in wicking contact with a sink.
  • the conjugate pad holds a supply of antibody specific for an epitope of the RSV fusion protein.
  • the antibody has been conjugated with finely divided colloidal gold particles, and the conjugate has been treated to be easily released from the conjugate pad when contacted with a liquid sample.
  • a capture antibody specific for a second epitope on the fusion protein has been immobilized on the test-reading membrane.
  • the capture antibody is preferably immobilized in a line traversing the expected flow path of sample along the test-reading membrane.
  • the preferred embodiment further includes a control signal that is a ligand capable of capturing the conjugate even when not bound to an antigen.
  • the control signal ligand is immobilized on the test-reading membrane downstream from the capture antibody.
  • the wicking member is supported on a backing that is, preferably, a single, non-absorbent piece to which the wicking member fixed with adhesive.
  • the present invention comprises a system for detecting RSV infections comprising the device described above and an extraction reagent for preparing samples for testing.
  • the extraction reagent includes a surfactant, an acid and a protein extraction reagent.
  • the present invention comprises an assay for RSV infection including the steps of obtaining a mucous sample from a patient, incubating the sample with an extraction reagent to form a test sample, applying the test sample to the device described above and determining whether a signal appears at the location of the capture antibody after a time.
  • FIG. 1 shows in perspective view a preferred embodiment of the wicking member.
  • FIG. 2 shows in plan view the components of the device that comprise the case.
  • FIG 3 shows in schematic view possible test results.
  • FIG. 1 shows the components of wicking member 1.
  • Adhesive carrier 2 is a single piece of non-absorbent vinyl coated with an adhesive to retain in position the other components.
  • Sample pad 3 is in wicking contact with conjugate pad 4.
  • Conjugate pad 4 is in wicking contact with test-reading membrane 5, which is in turn in wicking contact with sink 6.
  • Wicking member 1 has a width of about 6 mm and a total length of about 80 mm in a preferred embodiment .
  • Sample pad 3 of this embodiment is formed from commercially available paper and is treated with a solution of non-ionic surfactant and polyvinyl alcohol to block any attraction between the sample pad material and the fusion protein in the sample.
  • the sample pad is about 6 mm by about 19 mm.
  • Conjugate pad 4 is preferably about 6 mm by 6 mm in size.
  • conjugate pad 4 also a commercially available material, is loaded with a conjugate comprising an antibody against an epitope of the fusion protein that has been conjugated with colloidal gold particles.
  • an aqueous solution of the conjugate and a saccharide is added to the pad and lyophilized in place.
  • the conjugate- saccharide solution can be dried in place by heat.
  • Test-reading membrane 5 is made from commercially available nitrocellulose paper and is about 6 mm by about 25.4 mm in size. It is blocked as was sample application pad 3 and a stripe of an antibody specific for a second epitope on the fusion protein is immobilized on its surface. A stripe of goat anti-mouse immunoglobulin is also immobilized on membrane 5 to serve as a control. The preferred location for the control is further downstream in the expected path of the sample than the immobilized antibody stripe.
  • Sink 6 has a size of about 6 mm by about 37 mm and is formed from commercially available absorbent paper.
  • FIG. 2 shows in plan view components that make up the top and bottom of the case that holds wicking member 1 of FIG. 1.
  • Component 10 is the top of the case and component 20 is the bottom.
  • Component 10 includes sample receiving port 11, test port 12 and vents 13.
  • Sample receiving port 11 includes a periphery 14 connected by a downwardly sloping surface 15 to opening 16 positioned to provide access to sample pad 3 of wicking member 1.
  • opening 16 is of a generally rectangular shape having a long dimension of about 7.6 mm and a short dimension of about 3.8 mm while opening 16 is about 2.4 mm below periphery 14.
  • the volume 17 defined by periphery 14, surface 15 and opening 16 is, in the preferred embodiment, about 275 microliters.
  • Volume 17 is adequate to receive slightly in excess of the preferred sample size of about 230 microliters and to provide a head pressure that encourages movement of the sample into sample pad 3.
  • Volume 17 also effectively limits the size of the sample to a maximum of about 275 microliters. Medical professionals performing the assay can stop adding sample when volume 17 is filled.
  • test-reading port 12 positioned to expose to view both the immobilized antibody and the control described in connection with FIG. 1.
  • the test-reading port has a generally rectangular shape with a long side of about 11.5 mm and a short side of about 5.6 mm. Periphery 19 around port 12 is optional.
  • Vents 13 provide drying access to the atmosphere for liquid in sink 6. Vents 13 operate with volume 17 to enable the test device of the present invention to be used as a permanent record of the assay performed. Volume 17 effectively limits the amount of liquid sample entering the test device, and vents 13 encourage rapid drying of liquid reaching sink 6. This avoids backflow of liquid containing the conjugate into test-reading membrane 5 of liquid after it has reached sink 6. Such backflow is thought to be a contributor to assay results that change over time as described above.
  • Lower component 20 includes raised flanges 22 to maintain wicking member 1 in position under component 10 without pinching it or otherwise restricting the migration of liquid in it.
  • FIGS. 3a, 3b and 3c show schematically three possible assay results that may be observed in port 17.
  • FIG. 3a shows a positive result where conjugate bound to fusion protein has been captured and concentrated by the immobilized antibody to make it visible as stripe 31.
  • the control has captured conjugate, making it visible as control stripe 32 and indicating that the test has reached completion.
  • the result shown in FIG. 3a is an indication of fusion protein in the sample and of RSV infection in the patient providing the sample.
  • FIG. 3b shows a negative result.
  • the absence of a colored stripe at the "T” location indicates an absence of fusion protein in the sample.
  • the immobilized capture antibody at the "T” location which binds specifically to it, is not able to capture the conjugate.
  • the immobilized control at the "C” location captures conjugate in a concentration sufficient to make it visible, indicating that the test operated correctly and is complete.
  • FIG. 3c shows no result after a predetermined time (normally about 15 minutes) indicating only that the test did not operate properly.
  • FIGS. 2 and 3 were made for testing mucous samples obtained from 100 patients presenting symptoms similar to those of RSV infection. Mucous was obtained by aspiration or by nasal swab. A portion was set aside for confirmation testing and a portion was placed briefly in the extraction reagent described above, resulting in a reagent/sample combination. About 230 microliters of the combination was placed in volume 17 of the device, and the condition of test-reading port 18 was observed after 15 minutes. Results in port 18 as shown in FIG. 3a were listed as positive for RSV infection and results as shown in FIG 3b were listed as negative.
  • the used assay devices were retained and examined at 1 hour, 1 day, 1 week and 9 months for changes in results, including especially negative results that later became false positives and positive results that later faded or blurred. There were no changes.
  • the example indicates that the assay of the present invention is accurate for RSV infection ' and that the test device can act as a permanent record of the test results.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne une bande d'essai de dosage immunologique à écoulement latéral contenue dans un boîtier facilitant le contrôle de la dimension de l'échantillon et le séchage de l'échantillon liquide atteignant le puits de manière que les résultats présentés par un conjugué signal-anticorps capturé soient permanents, permettant d'utiliser le dispositif d'essai comme un enregistrement permanent des résultats de l'analyse.
PCT/US2002/039999 2001-12-14 2002-12-14 Dosage immunologique rapide du rsv WO2003052379A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002361677A AU2002361677A1 (en) 2001-12-14 2002-12-14 Rapid immunoassay for rsv

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US34023101P 2001-12-14 2001-12-14
US60/340,231 2001-12-14

Publications (2)

Publication Number Publication Date
WO2003052379A2 true WO2003052379A2 (fr) 2003-06-26
WO2003052379A3 WO2003052379A3 (fr) 2003-10-09

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WO (1) WO2003052379A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1767941A1 (fr) * 2004-06-07 2007-03-28 DENKA SEIKEN Co., Ltd. Appareil chromatographique de détection, procédé de test et kit utilisant celui-ci
WO2007122403A1 (fr) * 2006-04-21 2007-11-01 British Pregnancy Advisory Service Dispositif et PROCÉDÉ PERMETTANT la DÉTECTION d'une hormone ASSOCIÉE À la grossesse
CN101427137B (zh) * 2006-04-21 2012-08-29 英国孕育咨询服务公司 用于检测妊娠相关激素的装置和方法
AT513678A1 (de) * 2012-11-27 2014-06-15 Kim Scheuringer Testvorrichtung
WO2020233533A1 (fr) * 2019-05-17 2020-11-26 Mei Jun Pièce d'essai permettant la détection quantitative rapide de protéines de cellules tissulaires

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2684488A (en) * 1988-06-27 1990-01-04 Carter-Wallace, Inc. Test device and method for colored particle immunoassay
US5141850A (en) * 1990-02-07 1992-08-25 Hygeia Sciences, Inc. Porous strip form assay device method
US6258548B1 (en) * 1997-06-05 2001-07-10 A-Fem Medical Corporation Single or multiple analyte semi-quantitative/quantitative rapid diagnostic lateral flow test system for large molecules

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1767941A1 (fr) * 2004-06-07 2007-03-28 DENKA SEIKEN Co., Ltd. Appareil chromatographique de détection, procédé de test et kit utilisant celui-ci
EP1767941B1 (fr) * 2004-06-07 2010-11-24 DENKA SEIKEN Co., Ltd. Appareil chromatographique de détection
WO2007122403A1 (fr) * 2006-04-21 2007-11-01 British Pregnancy Advisory Service Dispositif et PROCÉDÉ PERMETTANT la DÉTECTION d'une hormone ASSOCIÉE À la grossesse
CN101427137B (zh) * 2006-04-21 2012-08-29 英国孕育咨询服务公司 用于检测妊娠相关激素的装置和方法
AT513678A1 (de) * 2012-11-27 2014-06-15 Kim Scheuringer Testvorrichtung
AT513678B1 (de) * 2012-11-27 2016-01-15 Kim Scheuringer Testvorrichtung
WO2020233533A1 (fr) * 2019-05-17 2020-11-26 Mei Jun Pièce d'essai permettant la détection quantitative rapide de protéines de cellules tissulaires

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Publication number Publication date
WO2003052379A3 (fr) 2003-10-09
AU2002361677A8 (en) 2003-06-30
AU2002361677A1 (en) 2003-06-30

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