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WO1996021863A1 - Kit de dosage immunologique et procede de detection rapide d'anticorps diriges contre vih-1 et vih-2 - Google Patents

Kit de dosage immunologique et procede de detection rapide d'anticorps diriges contre vih-1 et vih-2 Download PDF

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Publication number
WO1996021863A1
WO1996021863A1 PCT/US1995/002351 US9502351W WO9621863A1 WO 1996021863 A1 WO1996021863 A1 WO 1996021863A1 US 9502351 W US9502351 W US 9502351W WO 9621863 A1 WO9621863 A1 WO 9621863A1
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WO
WIPO (PCT)
Prior art keywords
membrane
hiv
antibodies
fluid
test device
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US1995/002351
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English (en)
Inventor
Robert Chen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
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
Priority claimed from PCT/US1995/000305 external-priority patent/WO1995018624A1/fr
Application filed by Individual filed Critical Individual
Priority to AU19701/95A priority Critical patent/AU1970195A/en
Priority to PE1995280131A priority patent/PE48196A1/es
Publication of WO1996021863A1 publication Critical patent/WO1996021863A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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
    • G01N33/56988—HIV or HTLV

Definitions

  • This invention relates to an immunoassay kit for rapid detection of HIV-1 and HIV-2 antibodies in biological fluid, and related method.
  • AIDS Abnormal Immunodeficiency Syndrome
  • T helper cells a subpopulation of T cells
  • the presence of the virus in the patient causes the immune system to elicit the production of anti-HIV-1 or HIV-2 antibodies.
  • Tests to detect these anti-HIV-1 or HIV-2 antibodies are now widely used in order to identify infected patients and for the screening of blood derived products.
  • the synthetic peptides gP41, a transmembrane glycoprotein derived from HIV-1 and gP36 derived from HIV-2 represent major antigenic sites for anti-HIV antibodies. Fragments of these proteins and other HIV antigenic transmembrane glycoproteins such as gP38 and gP120 have been synthetically produced in the laboratory with the help of advanced genetic engineering technologies by others and are commercially available under these designations.
  • the present invention is a rapid membrane- based immunodiagnostic assay for detection of HIV-1 and HIV-2 antibodies.
  • the assay utilizes HIV peptide antigens on the membrane surface which are immunoreactive with antibodies in the fluid sample that are specific to HIV-l/HIV-2. After reaction such antibodies are visualized with a Protein A colloidal gold conjugate resulting in the display of visible red or red like spot on the membrane at the immunoreaction locus. Absence of antibodies in the fluid is indicated by a absence of color on the membrane.
  • the present invention provides a test device and method for an accurate, rapid and simplified assay for detecting the presence of antibodies to HIV-1 and HIV-2. It has been found that this assay provides high sensitivity and high specificity in detecting these antibodies in a single test in many cases less than three minutes after drawing a human blood sample. This makes it suitable for immediate field testing, without the need for laboratory facility involvement, and can be performed routinely by trained non-medical personnel.
  • the test device for the detection of antibodies to HIV-1 and HIV-2 comprises a filter device segment and a reaction cell.
  • the filter device encases a filtration medium and is useful for removing red blood cells from a fluid such as a blood sample, if necessary, and is detachably attached to the reaction cell. Any filtered fluid such as serum can pass through the filter medium immediately into the reaction cell encountering the reaction membrane encased within the reaction cell.
  • the reaction cell membrane is coated with antigen immunoreactive to HIV-1 and HIV-2 antibodies within the fluid.
  • the filter device portion can be detached and discarded. After suitable treatment of the membrane which culminates in treatment of the membrane with an indicator such as Protein A colloidal gold conjugate, a red color may be viewed on the reaction cell membrane indicative of the presence of antibodies to HIV-1 or HIV-2.
  • the method comprises obtaining a fluid sample, filtering the fluid sample, if necessary, through the filter device separating the red blood cells from the sample, coating the test or reaction cell membrane with antigenic glycoproteins of gP41, gP36, gP38 and gP120, contacting the sample with the coated membrane to allow for the immunoreaction of any antibodies with the antigen, conjugating the antibodies with a Protein A colloidal color indicator, viewing the membrane for presence of a red color, and confirming the existence or nonexistence of the subject antibodies.
  • Fig. 1 exhibits the pouch, desiccant, pipette and fully assembled test device
  • Fig. 3 is a perspective view of the filter device separated from the reaction cell after filtration and immunoreaction and rotated to view the filter medium, the reaction cell membrane displaying a visually readable color indicating HIV antibody presence;
  • Fig. 4 is a cross-section view of the assembly of Fig. 2 taken along 4-4; selected dimensions in Figure 4 are:
  • the test device of the present invention can be used to screen a biological fluid sample for antibodies to HIV-1 and HIV-2.
  • the biological samples which can be so screened include but are not limited to whole blood or components such as serum or plasma. Other biological fluids may be used.
  • the test is used to screen whole human blood, serum, or plasma.
  • the kit is comprised of a testing device having a filter portion and reaction cell portion, a pipette, and appropriate solutions of Protein A gold colloidal conjugate, wash buffer, serum buffer, sealed in a pouch or container that is resistent to external moisture, with desiccant.
  • the assembled filter test device 1 is illustrated in Figure 1. It is comprised of a plastic filter device 2, having a filtration funnel opening 2A in which the fluid to be filtered is placed, said filter device 2 is detachably attached to a plastic reaction cell 3 which has an upper casing 4 and a lower casing 5.
  • the reaction cell 3 is assembled by friction fit of casings 4, 5.
  • the pipette 6 desiccant 7 and pouch 8 are also illustrated.
  • the filter device 2 is held in place by friction fit within the aperture 4A of the upper reaction cell casing 4 by friction contact of the three flanges 15, symmetrically disposed around the outer wall of the female funnel piece 14, with the interface 28 between the flanges 15 and the upper reaction cell casing wall 4.
  • the filter device can be removed from the reaction cell by the tab portion 12 (as illustrated in Figure 3) and discarded.
  • FIGS 3 through 6 provide greater detail of the filter device and reaction cell, when the filter device is either seated within the aperture of the reaction cell or removed as illustrated.
  • the filter device is comprised of three general parts: a male funnel piece 13, a female funnel piece 14 and the filter medium 19. These parts, once assembled in appropriate fashion as by compression assembly, are not intended to be routinely disassembled during or after use or operation.
  • the assembled filter device wherein the male funnel 13 and female funnel 14 pieces are mated (as illustrated in Figures 3 and 4) achieves several purposes.
  • the filter medium is comprised of two layers, pressed together, a top coarse layer 22 which is more porous and provides for more rapid filtration, and a bottom finer layer 23 for slower filtration. It is preferred that the finer layer 23 have porosity of about 122 cm 3 /s/cm 2 . While the smaller pore size slows the overall filtration time, it improves the subsequent reactivity between the fluid antibodies and the antigen coated membrane.
  • the coarse filter or upper filter 22 is comprised of 100% borosilicate glass fiber which is white and unfinished in appearance and has a basis weight of 88 g/m 2 , a thickness of 0.44 mm, a flow rate of 20 ml/min, air permeability of 1.4 cfm/ft 2 , and retention of 0.7 ⁇ m.
  • the lower filter or finer filter 23 is borosilicate glass fiber, also white, has a basis weight of 70 g/m 2 , a thickness of .381 mm, a tensile machine direction of 6.7 kg/25mm, a tensile cross direction of 3.6 kg/25mm, a tear machine direction of 155 g., a porosity of 122 cm'/s/cm 2 , and ash percentage of 95%.
  • the filters can be obtained from Ahlstrom Filtration in Mount Holly Springs, Pennsylvania, and are designated as Grade 151 and .015" Manninglas 1200, respectively.
  • the male piece 13 and female piece 14 are compressed to fit along their interface 27A, which is between wall 17 of the male piece 13 and wall 27 of the female piece 14. Each of these walls terminates in the collar portions 21 and 29, respectively.
  • the filter device is snugly fit within the upper reaction cell casing 4 such that the bottom filter layer 23 of the filter medium is in substantial contact with the reaction cell membrane 10, as illustrated in Figure 4.
  • Figure 5 illustrates an exploded view of the reaction cell.
  • Reaction cell 3 has an upper casing 4 and a lower casing 5. Inside the lower casing 5 is a reservoir 34 bounded by wall 33. Inside the reservoir 34 sits an absorbent cotton pad 31, which is in substantial surface contact with porous adhesive gauze backing 30, which backing fixes the membrane 10 to a hard plastic panel 11, which panel has a reaction aperture 35 centrally positioned to directly contact and engage the lower filter layer 23 within aperture 35. This small opening or aperture 35 on the panel 11 exposes a localized portion of the membrane 10 to the assayed fluid.
  • the vertical sequence, as illustrated in Figures 1, 3, and 4, of filter medium/membrane/backing/absorbent pad provides a path of continuous contact of absorbent materials for enhanced fluid diffusion and absorption.
  • the membrane is comprised of nitrocellulose paper which is commercially available from Schleicher and Schuell GmbH. It can be first cut into suitably sized pieces for preparation and can be as small as necessary to fit within the dimensions of the test device, or can be reduced to appropriate size after membrane coating.
  • the membrane is preferably prepared as follows: gP41 is dissolved in 50mM sodium carbonate/bicarbonate (pH 9.3) to a final concentration of 20 ⁇ g/ml, and the nitrocellulose membrane is allowed to soak within the solution for about 2 hours at room temperature to coat the membrane. Then the membrane is air dried or vacuum dried. The membrane is then soaked in a similar solution of dissolved gP36 and appropriately dried in the same manner as well. Finally, the membrane is soaked in a solution of gP38 and gP120 in a ratio of about three parts (micrograms/milliliter) gP38 to one part gP120 prepared in the same fashion, and then dried in the same fashion.
  • transmembrane glycoproteins gP 41, 36, 38 and 120 are commercially available and known by these designations.
  • the complete nucleotide sequence of glycoproteins are listed in Nature 1985, 313:277-284 by Ratner, I. et al. It is preferred that each glycoprotein be at least 95% pure. In the event that any glycoprotein is not pure enough, then prior to coating it should be purified by any suitable means such as column chromatography.
  • the reaction cell membrane surface area of the nitrocellulose membrane is accessible for further treatment and can be viewed, as illustrated in Figure 3.
  • Two drops of conventional serum buffer comprised of Tris buffer and protein stabilizer, are added directly to the membrane.
  • two drops of conventional wash buffer comprised of Tris buffer, are added to the membrane to eliminate any discoloration of any kind such as that caused by ruptured or lysed red blood cells and hemoglobin discoloration.
  • two drops of Protein A gold colloidal conjugate are added.
  • two more drops of wash buffer are added. Shortly thereafter the red spot will appear, as illustrated in Figure 3, or will not appear on the membrane. The presence of the red spot indicates the positive identification of antibodies to HIV-1 and/or HIV-2.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (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)
  • AIDS & HIV (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Dispositif de dosage immunologique destiné à la détection d'anticorps dirigés contre VIH-1 et VIH-2 dans des liquides biologiques en vue d'une réaction immunologique immédiate et de la détection de la présence desdits anticorps, qui comprend un dispositif filtre et une cellule de réaction assemblés, ledit dispositif filtre étant détachable de la cellule de réaction, une fois la filtration achevée. Ladite cellule de réaction comprend une membrane sur laquelle ou dans laquelle se produit la réaction immunologique. Une fois la réaction terminée et le filtre enlevé, si nécessaire, il est possible d'observer ladite membrane et en particulier l'indicateur pour déterminer la présence ou l'absence d'anticorps. Ledit dosage peut également être effectué directement sur la membrane sans utiliser le filtre. Un procédé associé au dosage immunologique d'anticorps contre VIH-1 et VIH-2 est également décrit.
PCT/US1995/002351 1995-01-09 1995-02-23 Kit de dosage immunologique et procede de detection rapide d'anticorps diriges contre vih-1 et vih-2 Ceased WO1996021863A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU19701/95A AU1970195A (en) 1995-01-09 1995-02-23 Immunodiagnostic kit and method for rapid detection of HIV-1 and HIV-2 antibodies
PE1995280131A PE48196A1 (es) 1995-02-23 1995-09-27 Estuche para inmunodiagnostico y metodo para deteccion de anticuerpos hiv-1 y hiv-2

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/US1995/000305 WO1995018624A1 (fr) 1994-01-10 1995-01-09 Evaluation du test red-dot de vih 1 et vih 2 pour la detection d'anticorps anti vih 1 et anti vih 2
USPCT/US95/00305 1995-01-09

Publications (1)

Publication Number Publication Date
WO1996021863A1 true WO1996021863A1 (fr) 1996-07-18

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PCT/US1995/002351 Ceased WO1996021863A1 (fr) 1995-01-09 1995-02-23 Kit de dosage immunologique et procede de detection rapide d'anticorps diriges contre vih-1 et vih-2

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AU (1) AU1970195A (fr)
WO (1) WO1996021863A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004040306A1 (fr) * 2002-10-31 2004-05-13 Labrea Holding S.A. Dispositif de diagnostic et kit
WO2006047869A1 (fr) 2004-11-01 2006-05-11 International Bio-Therapeutic Research Inc. Systeme de tests immunodiagnostiques jetables
US8475735B2 (en) 2004-11-01 2013-07-02 Uma Mahesh Babu Disposable immunodiagnostic test system

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3791933A (en) * 1971-02-25 1974-02-12 Geomet Rapid methods for assay of enzyme substrates and metabolites
US5008080A (en) * 1985-10-04 1991-04-16 Abbott Laboratories Solid-phase analytical device and method for using same
US5039604A (en) * 1987-08-21 1991-08-13 Cellular Products, Inc. Test device and method of preparing same, assay kit and method for the simultaneous detection of two HTLV or HIV antibodies
US5096809A (en) * 1988-07-25 1992-03-17 Pacific Biotech, Inc. Whole blood assays using porous membrane support devices
US5160701A (en) * 1986-02-18 1992-11-03 Abbott Laboratories Solid-phase analytical device and method for using same
US5166051A (en) * 1990-08-08 1992-11-24 Genesis Labs, Inc. Membranes, membrane overlays for exclusion of erythrocytes, and method for immunoassay of whole blood analytes
US5260189A (en) * 1988-12-20 1993-11-09 Immunodiagnostics, Inc. Synthetic HIV-like peptides their compositions and uses

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3791933A (en) * 1971-02-25 1974-02-12 Geomet Rapid methods for assay of enzyme substrates and metabolites
US5008080A (en) * 1985-10-04 1991-04-16 Abbott Laboratories Solid-phase analytical device and method for using same
US5160701A (en) * 1986-02-18 1992-11-03 Abbott Laboratories Solid-phase analytical device and method for using same
US5039604A (en) * 1987-08-21 1991-08-13 Cellular Products, Inc. Test device and method of preparing same, assay kit and method for the simultaneous detection of two HTLV or HIV antibodies
US5096809A (en) * 1988-07-25 1992-03-17 Pacific Biotech, Inc. Whole blood assays using porous membrane support devices
US5260189A (en) * 1988-12-20 1993-11-09 Immunodiagnostics, Inc. Synthetic HIV-like peptides their compositions and uses
US5166051A (en) * 1990-08-08 1992-11-24 Genesis Labs, Inc. Membranes, membrane overlays for exclusion of erythrocytes, and method for immunoassay of whole blood analytes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF ACQUIRED IMMUNE DEFICIENCY SYNDROMES, Volume 2, Number 2, issued April 1989, MODROW et al., "Carrier Bound Synthetic Oligopeptides in ELISA Test Systems for Distinction Between HIV-1 and HIV-2 Infection", pages 141-148. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004040306A1 (fr) * 2002-10-31 2004-05-13 Labrea Holding S.A. Dispositif de diagnostic et kit
WO2006047869A1 (fr) 2004-11-01 2006-05-11 International Bio-Therapeutic Research Inc. Systeme de tests immunodiagnostiques jetables
AU2005301045B2 (en) * 2004-11-01 2012-10-18 International Bio-Therapeutic Research Inc. Disposable immunodiagnostic test system
US8475735B2 (en) 2004-11-01 2013-07-02 Uma Mahesh Babu Disposable immunodiagnostic test system

Also Published As

Publication number Publication date
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