EP0438457A1 - Procede de detection simultanee de differents types d'anticorps et/ou d'antigenes produits par des cellules individuelles - Google Patents

Procede de detection simultanee de differents types d'anticorps et/ou d'antigenes produits par des cellules individuelles

Info

Publication number
EP0438457A1
EP0438457A1 EP89911411A EP89911411A EP0438457A1 EP 0438457 A1 EP0438457 A1 EP 0438457A1 EP 89911411 A EP89911411 A EP 89911411A EP 89911411 A EP89911411 A EP 89911411A EP 0438457 A1 EP0438457 A1 EP 0438457A1
Authority
EP
European Patent Office
Prior art keywords
antibodies
antigens
detected
different types
different
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.)
Withdrawn
Application number
EP89911411A
Other languages
German (de)
English (en)
Inventor
Anders Vahlne
Cecil Czerkinsky
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.)
Syntello AB
Original Assignee
Syntello AB
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 SE8803689A external-priority patent/SE465943B/sv
Application filed by Syntello AB filed Critical Syntello AB
Publication of EP0438457A1 publication Critical patent/EP0438457A1/fr
Withdrawn legal-status Critical Current

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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • Tne present invention refers to a method for detection o antibodies and/or antigens secreted and/or released by individua cells, at which a cell suspension is brought in contact with solid carrier, and after that or simultaneously therewith othe antibodies are added which are directed against and have a ability to bind to the antibodies or antigens that are to b detected, and which other antibodies are provided with an enzym reacting with an indicator substance for detecting the antibodie or antigens in question.
  • Laboratory diagnosis of e.g. virus infections is mainly based o either detection of the infection-inducing virus itself or part thereof, so called antigens, in samples from patients, or whic is more common on indication of antibodies in the patient' blood directed against the virus.
  • a co ⁇ u ⁇ on method for antibod detection is ELISA ("enzyme linked immunosorbent assays")
  • ELISPOT enzyme linked immunosorbent assays
  • the object of the present invention is to provide a method base on ELISPOT, by means of which it is possible to detect simul taneously at least two types of antibodies and/or antigens.
  • Th;. has according to the invention been achieved by the fact that a least two types of said other antibodies are added directe against at least two different types of antibodies that are to b detected and which other antibodies are provided with differen enzymes, and that then the corresponding number of indicato substances are added having different staining effect, fo simultaneous detection of the different types of antibodie and/or antigens by evaluation of spots of distinct colour remaining on the solid carrier.
  • a combination of the two abov mentioned indicator systems in the form of AP- and HRP- labelle antibodies and corresponding chromogen substrate were used fo simultaneous detection of distinct types of antibody-producin cells.
  • zones of IgG and IgA antibodie bound to a solid carrier and produced by distinct cells were visualized in the form of blue and red spots resp. correspondin to either of these different types of antibodies.
  • PBMC Peripheral blood mononuclear cell
  • Interface PBMC were washed twice with isotoni phosphate-buffered saline (0.01 M phosphates, 0.15 M NaCl, pH 7.4) (PBS), and resuspended at the appropriate densities in assay culture medium.
  • PBS isotoni phosphate-buffered saline
  • FBS foetal bovine serum
  • AP- or HRP-cojugated affinity-purified goat anti-human IgA and goat anti-human IgG anti-bodies were purchased commersially.
  • the AP chromogen substrate soultion consisting of 5-bromo-4-chloro- 3-indolyl phosphate toluidine salt and p-nitroblue tetrazolium chloride (BCIP/NBT) was prepared according to the manufacturer's instructions (Bio-Rad Laboratories, Richmond, CA).
  • the assay consists of five stages: 1) first, a solid phase immun ⁇ adsorbent is prepared;
  • the standard ELISPOT assay was modified by using nitrocellulose membranes as the solid support instead of polystyrene.
  • Individual wells of nitrocellulose bottomed 96-well Millititer HA plates (Millipore, Bedford, MA) were filled with 0.075-0.1 ml of PBS containing 0.2 ⁇ g of influenza virus hemaggultinin ( yett Laboratories) . Unadsorbed proteins were removed by three successive manual washings with PBS and the plates were immersed in this buffer for 5 min. Wells were the emptied of wash buffer and the outer surface of the nitrocellulose membrane was carefully dried with absorbent paper towels.
  • the content of the wells was replaced with 0.1 ml of cell suspensions containing various numbers of PBMC. Routinely, we used at least three sets of triplicate wells. Each set of wells recieved 2 x 10 5 , 10 5 and 5 x 10 4 PBMC/well. Plates were then incubated undisturbed for 3-4 h at 37°C in a CO2 incubator. In one experiment, PBMC were incubated for 5 h at 37 ⁇ C with various concentrations (5 x 10 ⁇ 4 M, 10" ⁇ M, 2 x 10 ⁇ 3 M) 0 f cycloheximide (Sigma) in assay culture medium, washed, resupended and plated in cycloheximide containing medium.
  • Concentrations ranging from 0.5 ⁇ g/ml to 2.5 ⁇ g/ml were used for both types of enzyme conjugate. Plates were incubated for 3 h at room temper ⁇ ature or overnight at 4 ° C . Dishes were then rinsed four times with PBS and immersed in 0.05 M Tris buffer saline, pH 8.0, for 5 min prior to development.
  • Peripheral blood PBMC were obtained from one volunteer 7 day after immunization with influenza virus vaccine. Values ar expressed as spot-forming cells (SFC) numbers of quadroplicat assay wells. Table 1
  • PBMC-secreting IgG antibodies and PBMC- secreting IgA antibodies to influenza virus could be detected simultaneously in all four volunteers examined.
  • ASC were detected as early as 5 days after systemic immunization with influenza virus vaccine.
  • Spot forming cell (SFC) numbers reached a maximum on day 7, by day 9-12 the frequency of virus- specific SFC markedly decreased (data not illustrated).
  • Influenza virus specific IgG-SFC and IgA-SFC responses followed a similar kinetic pattern but differed in magnitude.
  • the specifity of the assay for simultaneous demonstration o influenza virus specific-IgA ASC and IgG ASC was documented b several observations.
  • PBMC Peripheral blood PBMC were obtained from one donor on day 7 following immunization with influenza virus vaccine. PBMC were assayed by two-colour ELISPOT assay for numbers of virus-secreting ASC.
  • IgG SFC and IgA SFC were developed with AP-conjugated anti-IgG and HRP-conjugated anti-IgA, respectively followed by BCIP/NBT (blue) and AEC/H 2 02 (red) enzyme substrates. Values represent mean SFC numbers of quadroplicate assay wells/10 5 PBMC. Data in parentheses indicate percentages of inhibition.
  • Inhibitor added SFC numbers/10 6 PBMC per assay well IgG (blue) (IgA) red
  • the potential of the method according to the invention has been confirmed for detecting simultaneously cells secreting other Ig isotypes (IgM, IgG subclasses) in both human and murine systems.
  • the method should also be applicable for detection of other types of antibody-producing cells, e.g. lymphokine-secreting cells, and for detection of antigens, i.e. the virus itself or parts thereof, and for simultaneous detection of antibodies and antigens.
  • the antibodies or antigens to be detected can be of eucarytic, bacterial, viral or parasitic origin.
  • the receptors e.g. antigens or antibodies (in case antigens are to be detected) bound to the carrier can be of one single type reacting with the different types of antibodies or antigens resp. to be detected. They can also be of different types reacting with a type each of antibodies and/or antigens to be detected.
  • the solid carrier can also consist of two opposed plates, at which one type of antigens or antibodies is bound to one of the plates and another of antigens or antibodies is bound to the opposed plate, at which the cell suspension is applied between the plates. It is in this way possible to detect simultaneously several types of antigens and/or antibodies. For certain substances, e.g.
  • the solid carrier does not have to have antigens or anitbodies bound thereto, since these substances can bind directly to the solid carrier, in case this is of a material having intrinsic bindning properties, e.g. nitro- cellulose, nylon or polyvinyl.

Landscapes

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

Abstract

Dans le procédé décrit, qui sert à la détection d'anticorps et/ou d'antigènes sécrétés et/ou libérés par des cellules individuelles, une suspension de cellules est mise en contact avec un support solide. Après ou pendant cette opération de mise en contact, on ajoute au moins deux types différents d'autres anticorps dirigés contre respectivement les anticorps ou les antigènes à détecter et ayant la capacité de se lier respectivement à ces anticorps ou à ces antigènes à détecter, ces autres anticorps étant en outre dotés d'enzymes différentes. On ajoute ensuite le nombre correspondant de substances indicatrices, lesquelles ont un effet colorant différent pour la détection simultanée des différents types d'anticorps et/ou d'antigènes par évaluation des tâches de couleurs distinctes restant sur le support solide. Selon le mode de réalisation décrit, on utilise deux supports solides, aux surfaces opposées desquels sont attachés des antigènes et/ou des anticorps du même type ou d'un type différent, la suspension de cellules étant appliquée entre ces surfaces.
EP89911411A 1988-10-14 1989-10-13 Procede de detection simultanee de differents types d'anticorps et/ou d'antigenes produits par des cellules individuelles Withdrawn EP0438457A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
SE8803689 1988-10-14
SE8803689A SE465943B (sv) 1988-10-14 1988-10-14 Metod foer samtidig detektering av olika analyter, spec antikroppar med "elispot" tekniken
US36737789A 1989-06-16 1989-06-16
US367377 1989-06-16

Publications (1)

Publication Number Publication Date
EP0438457A1 true EP0438457A1 (fr) 1991-07-31

Family

ID=26660325

Family Applications (1)

Application Number Title Priority Date Filing Date
EP89911411A Withdrawn EP0438457A1 (fr) 1988-10-14 1989-10-13 Procede de detection simultanee de differents types d'anticorps et/ou d'antigenes produits par des cellules individuelles

Country Status (4)

Country Link
EP (1) EP0438457A1 (fr)
KR (1) KR900702371A (fr)
AU (1) AU4346989A (fr)
WO (1) WO1990004182A1 (fr)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8921880D0 (en) * 1989-09-28 1989-11-15 Medical Res Council Improvements in or relating to hybridomas
DE4211108A1 (de) * 1992-04-03 1993-10-07 Boehringer Mannheim Gmbh Viruslösung zum Einsatz bei Immunoassays
DE4232073C2 (de) * 1992-09-25 2001-09-20 Chiron Behring Gmbh & Co Verfahren zur immunchemischen Quantifizierung von inaktivierten, immunreaktiven Antigenen
DE4236189A1 (de) * 1992-10-27 1994-04-28 Boehringer Mannheim Gmbh Verfahren zur simultanen Bestimmung von Antigenen und Antikörpern
US5750356A (en) 1996-05-31 1998-05-12 Anergen, Inc. Method for monitoring T cell reactivity
DE19821289A1 (de) * 1998-05-13 1999-11-18 Volkmar Schoellhorn Verfahren zur Bestimmung der Aktivität von menschlichen und tierischen Zellen, insbesondere Blutzellen, und Mikrotiterplatten
EP1081496A1 (fr) * 1999-08-31 2001-03-07 Becton, Dickinson and Company Essai au passage continu de fluide pour la détection visuelle de la présence de l'influenza A et B
DE10125730A1 (de) * 2001-05-17 2002-11-21 A I D Autoimmun Diagnostika Gm Verfahren zum Nachweis von Erregern und durch diese induzierten zellulär produzierten Stoffen im lebenden Organismus
GB0409775D0 (en) 2004-04-30 2004-06-09 Mabtech Ab Assay
GB0409771D0 (en) 2004-04-30 2004-06-09 Mabtech Ab Assay
US20100221755A1 (en) * 2007-06-15 2010-09-02 University Of Rochester Use of antibody secreting cell elispot to assess antibody responses following antigen exposure
DE102007052517A1 (de) 2007-10-29 2009-04-30 Autoimmun Diagnostika Gmbh ELISPOT-Verfahren mit zwei Filtersystemen
GB0815675D0 (en) 2008-08-28 2008-10-08 Mabtech Ab Antibody secreting cell elispot

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1148859A (fr) * 1979-06-14 1983-06-28 Lacy R. Overby Technique pour le depistage simultane de deux virus de l'hepatite dans un milieu solide
US4690890A (en) * 1984-04-04 1987-09-01 Cetus Corporation Process for simultaneously detecting multiple antigens using dual sandwich immunometric assay
GB8607101D0 (en) * 1986-03-21 1986-04-30 Serono Diagnostics Ltd Immunoassay
WO1988005536A1 (fr) * 1987-01-20 1988-07-28 Interferon Sciences, Inc. Technique ''western blot'' multicolore

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9004182A1 *

Also Published As

Publication number Publication date
WO1990004182A1 (fr) 1990-04-19
AU4346989A (en) 1990-05-01
KR900702371A (ko) 1990-12-06

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