WO1986007633A1 - Test visant a determiner les reactions positives fausses dans une procedure d'analyse destinee a detecter la presence d'anticorps de micro-organismes - Google Patents

Test visant a determiner les reactions positives fausses dans une procedure d'analyse destinee a detecter la presence d'anticorps de micro-organismes Download PDF

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Publication number
WO1986007633A1
WO1986007633A1 PCT/US1986/001242 US8601242W WO8607633A1 WO 1986007633 A1 WO1986007633 A1 WO 1986007633A1 US 8601242 W US8601242 W US 8601242W WO 8607633 A1 WO8607633 A1 WO 8607633A1
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WO
WIPO (PCT)
Prior art keywords
test
cell line
microorganisms
infected
bound
Prior art date
Application number
PCT/US1986/001242
Other languages
English (en)
Inventor
Daniel H. Zimmerman
Sean P. O'neill
Gerald A. Bush
Judith A. Britz
Original Assignee
Electro-Nucleonics, Inc.
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 Electro-Nucleonics, Inc. filed Critical Electro-Nucleonics, Inc.
Publication of WO1986007633A1 publication Critical patent/WO1986007633A1/fr

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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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • 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
    • 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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
    • 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
    • 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

  • TITLE TEST FOR DETERMINING FALSE POSITIVE REACTIONS IN A TESTING PROCEDURE TO DETECT THE PRESENCE OF ANTIBODY TO MICROORGANISMS.
  • This invention relates to the determination of the presence of antibody to microorganisms. It has particular application for use with a highly sensitive test that determines the presence of antibody, particularly to eliminate false positive reactions.
  • Electro- Nucleonics, Inc. of Fairfield, New Jersey, U.S.A., has developed an Enzyme Linked Immunosorbent Assay (ELISA) test kit for identification of antibodies to the Human T- Lymphotropic Virus Type-Ill (HTLV-III).
  • ELISA Enzyme Linked Immunosorbent Assay
  • the present invention has particular application to the identification of false positives which may be due to poor sample quality, differences in laboratory environment/technique or reaction with cellular material and other biological/chemical substances used in the manufacture of the VIRGO test kit.
  • a specificity test to determine false positive reactions is developed from the same cell line used in the primary antibody-determining VIRGO test, but which is non-infected with the antigen of interest.
  • a specificity test component is prepared from a concentrate of cellular material shed by uninfected H9 cells, the cell line used in the growth of the HTLV-III virus used in the VIRGO test kit. (The H9 cell line is described in the Popovic et al. article cited below. ) The concentrate is treated in a manner idencial to that used for the propagation of the HTLV-III coated microassay plates in the VIRGO test kit.
  • a ratio of the absorbance values on each plate distingiushes a specific from a non-specific antibody reaction on HTLV-III, and false positive indications are detected.
  • the VIRGO test kit marketed by Electro-Nucleonics, Inc. utilizes HTLV-III isolated in the laboratory of Dr. Robert Gallo (National Cancer Institute) and propagated from a seed stock according to procedures established by M. Popovic (Popovic, M. , M. G. Sarngadharan, E. Read, and R.C. Gallo, 1984. "Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and Pre-AIDS.” Science 224, 497-500.).
  • the cell line infected with HTLV-III is cultured and the culture supernatant is purified by centrifugation procedures.
  • the viral concentrate is then inactivated in a two-step procedure using chemical and physical treatments. Purified inactivated HTLV-III is absorbed onto wells of a microassay plate to complete the VIRGO test kit.
  • HTLV-III microassay plate serum or plasma samples diluted in a buffer are added to the HTLV-III microassay plate. If antibodies specific for HTLV-III are present in a sample under test, they will form stable complexes with the HTLV-III antigens on the plate. A goat anti-human IgG (Heavy and Light chain specific) labeled with horseradish peroxidase is added. If the antigen/antibody complex is present, the peroxidase conjugate will bind and remain in the well. Enzyme substrate is then added. Color will develop in wells containing antibody. No color develops in negative wells. An acid stop solution is added to each well and the color read on a microassay plate reader at 492 nm.
  • IgG Heavy and Light chain specific
  • CJDSTSTUTE SHEET Specificity test plates in accordance with the present invention are prepared by adsorbing a concentrate of cellular material shed by uninfected H9 cells, the same cell line used in the growth of the HTLV-III virus in producing the VIRGO test kit.
  • the H9 concentrate is treated in a manner identical to that used for the preparation of the HTLV-III coated microassay plates in the VIRGO test kit.
  • H9 uninfected and HTLV-III infected cells are separately grown each as a standard suspension culture of between 2
  • the cells are re-fed every 2 - 3 days with an equal volume of fresh media of the same composition. In many cases this is accomplished by removing an equal volume of spent medium-containing cells.
  • the cells are normally removed by relative low speed (1000 xg) centrifugation. This so-called clarified extra ⁇ cellular fluid is then processed, or stored, at low temperature (+4 C or -20 C) until further processing.
  • the two samples are processed identically (insofar as possible).
  • the particular processing involves the use of a continuous flow centrifuge (RK).
  • the clarified fluid is pumped in at a constant flow rate and effluent is also removed at a similar flow rate.
  • the material (infected or uninfected) is banded in a sucrose density gradient. At the end of the run, the gradient is displaced by pumping 55% sucrose into the rotor. The gradient is collected in 25 - 30 equal volume fractions. The gradient is monitored
  • TE SHEET by use of a U.V. spectrophotometer recorder and also the density of each fraction is recorded.
  • the relevant fractions containing the viral materials are pooled, diluted and an equal volume of buffer to reduce the density and viscosity of the sucrose solution.
  • the virus is pelleted by high speed centrifugation and resuspended in Buffer devoid of sucrose. The material is then inactivated by the use of detergent and heat, samples removed for various quality control tests, aliquoted and stored at -70°C until use.
  • the uninfected fluid is identically processed and has all the same treatments and testing as described above for the infected fluid.
  • infected and uninfected materials are then coated onto solid supports by use of various buffers and then are packaged awaiting use in an assay.
  • the two materials infected primary test and non-infected specificity test are used identically in an immunoassay.
  • a light absorbance value of less than 0.100 indicates that the sample is nonreactive to HTLV-III antigen(s). If the detected absorbance indicates a possible positive reaction under the VIRGO procedure, the positive VIRGO test is preferably repeated and the sample is also identically processed using the H9 test plate. The VIRGO and H9 plate absorbances are then compared (ratio r) as follows:
  • a 1 and a_ are the detected light absorbances of the HTLV-III VIGRGO test plate and H9 specificity test plate, respectively, and K is a constant representing the intercept of the straight-line curve that results from a plotting of a 1 versus a terrain.
  • K is a constant representing the intercept of the straight-line curve that results from a plotting of a 1 versus a terrain.
  • k has been found to be 0.1.
  • the invention involves the use of a specificity test component prepared by using non-infected material of the same type as that used in producing infected materials for a primary test.
  • the invention obviously has application to other than the specific test procedure described above as the preferred embodiment of this invention.
  • soluble supports to which infected and uninfected materials are bound could be employed, e.g., soluble polymers which are polymerizable to complete detection, as in U.S. Patent No. 4,511,478.
  • testing criteria other than light absorbance as
  • SUBSTITUT SHEET in the above preferred embodiment could be employed, such as light reflectance, fluorescence, chemiluminescence, a precipitation pattern, to name some examples.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • AIDS & HIV (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Les réactions positives fausses détectées dans un test très sensible visant à déterminer la présence d'anticorps de micro-organismes se distingue des réactions positives vraies par l'utilisation d'un test de contrôle de la spécificité. Le test conduisant à la réaction positive devant être examinée utilise une fraction exempte de cellules d'une lignée cellulaire infectée de micro-organismes. Une fraction exempte de cellules de la dite lignée de cellules non infectée de micro-organismes est utilisée dans le test de contrôle de la spécificité afin de détecter les réactions positives fausses. Les résultats des tests utilisant des fractions exemptes de cellules infectées et non infectées sont comparées afin de distinguer les réactions positives fausses des réactions positives vraies.
PCT/US1986/001242 1985-06-17 1986-06-04 Test visant a determiner les reactions positives fausses dans une procedure d'analyse destinee a detecter la presence d'anticorps de micro-organismes WO1986007633A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US74576085A 1985-06-17 1985-06-17
US745,760 1985-06-17

Publications (1)

Publication Number Publication Date
WO1986007633A1 true WO1986007633A1 (fr) 1986-12-31

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PCT/US1986/001242 WO1986007633A1 (fr) 1985-06-17 1986-06-04 Test visant a determiner les reactions positives fausses dans une procedure d'analyse destinee a detecter la presence d'anticorps de micro-organismes

Country Status (3)

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EP (1) EP0227748A1 (fr)
AU (1) AU5990286A (fr)
WO (1) WO1986007633A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0243655A2 (fr) * 1986-03-19 1987-11-04 Roche Diagnostics GmbH Procédé et réactif pour la détermination d'un partenaire réactionnel d'une réaction immunologique
EP0311492A2 (fr) * 1987-09-30 1989-04-12 Elf Sanofi Trousse et méthode de dosage immunométrique applicables à des cellules entières
US6461825B1 (en) 1987-09-30 2002-10-08 Sanofi (Societe Anonyme) Immunometric assay kit and method applicable to whole cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0138667B1 (fr) * 1983-09-15 1991-11-21 INSTITUT PASTEUR Fondation reconnue d'utilité publique Méthode pour le diagnostic de lymphadénopathie et du syndrome d'immuno-dépression acquise

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0138667B1 (fr) * 1983-09-15 1991-11-21 INSTITUT PASTEUR Fondation reconnue d'utilité publique Méthode pour le diagnostic de lymphadénopathie et du syndrome d'immuno-dépression acquise

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Journal of The American Medical Association, Volume 253, Number 23, issued 21 June 1985, J.R. CARLSON et al., "Aids Serology Testing in Low- and High-Risk Group" see pages 3405-3408, see especially page 3408. *
New Zealand Medical Journal, Volume 98, Number 791, issued 27 November 1985, D. FIELD et al, "Assesment of HTLV-III Screening Tests in Christchurdi", see pages 992-3, see especially page 993 *
South African Medical Journal, Volume 68, issued 21 December 1985 L.V. MILNER et al, "Anti-HTLV-III Testing-a Practical Solution for Blood Transfusion Services?", see pages 921-2 see especially page 921. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0243655A2 (fr) * 1986-03-19 1987-11-04 Roche Diagnostics GmbH Procédé et réactif pour la détermination d'un partenaire réactionnel d'une réaction immunologique
EP0243655A3 (fr) * 1986-03-19 1991-01-16 Roche Diagnostics GmbH Procédé et réactif pour la détermination d'un partenaire réactionnel d'une réaction immunologique
EP0311492A2 (fr) * 1987-09-30 1989-04-12 Elf Sanofi Trousse et méthode de dosage immunométrique applicables à des cellules entières
EP0311492A3 (en) * 1987-09-30 1989-05-31 Sanofi, Societe Anonyme Kit and immunoassay method applicable to whole cells
US6461825B1 (en) 1987-09-30 2002-10-08 Sanofi (Societe Anonyme) Immunometric assay kit and method applicable to whole cells

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Publication number Publication date
EP0227748A1 (fr) 1987-07-08
AU5990286A (en) 1987-01-13

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