WO1999039209A1 - Dosage immunologique et trousse d'essai pour la determination d'une proteine fucosylee dans un echantillon biologique - Google Patents

Dosage immunologique et trousse d'essai pour la determination d'une proteine fucosylee dans un echantillon biologique Download PDF

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Publication number
WO1999039209A1
WO1999039209A1 PCT/EP1999/000639 EP9900639W WO9939209A1 WO 1999039209 A1 WO1999039209 A1 WO 1999039209A1 EP 9900639 W EP9900639 W EP 9900639W WO 9939209 A1 WO9939209 A1 WO 9939209A1
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WO
WIPO (PCT)
Prior art keywords
lectin
protein
fuc
agglutinin
labeled
Prior art date
Application number
PCT/EP1999/000639
Other languages
German (de)
English (en)
Inventor
Martin Holtzhauer
Sergej Ovodov
Alexander Knoll
Original Assignee
Biogenes Gmbh
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 Biogenes Gmbh filed Critical Biogenes Gmbh
Priority to JP2000529612A priority Critical patent/JP2002502037A/ja
Priority to EP99904839A priority patent/EP1053476A1/fr
Publication of WO1999039209A1 publication Critical patent/WO1999039209A1/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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57469—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving tumor associated glycolinkage, i.e. TAG
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57476—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncofetal proteins

Definitions

  • the invention relates to a sandwich immunoassay for determining the amount of fucosylated protein (Fuc protein) in a biological sample and associated reagent sets.
  • the invention relates to an immunoassay for the determination of fucosylated ⁇ -fetoprotein (AFP), which is important in the early detection of hepatocellular carcinoma (HCC) as a common form of cancer.
  • AFP fucosylated ⁇ -fetoprotein
  • Glycoproteins are recognized as tumor-associated. For example, in Aoyagi, Q. et al. (1985) Biochim. Biophys. Acta ⁇ 3C_, 217-223 and Aoyagi, Y. et al. (1988) Cancer
  • fucosylation of the pathological AFP as specific for hepatocellular carcinoma.
  • the determination of the amount of fucosylated protein can also be used when evaluating recombinant Glycoproteins produced, which are to be used as therapeutic agents, play a role, in particular in process control.
  • ConA Concanavalin A
  • LCA Lens-culinaris-Lectin A
  • PHA -E Phaseol us-vulgaris hemagglutinin E
  • the object of the invention was therefore to provide a detection method for fucosylated proteins which is sufficiently sensitive, specific and reproducible and can be used in clinical practice without problems.
  • the detection method should be easy to use and also suitable for routine examinations in the clinical area.
  • the immunoassay according to the invention for determining the amount of fucosylated protein in a biological sample is characterized in that a) an immobilized anti-fuc protein antibody or an immobilized anti-fuc protein antibody fragment with the biological sample to form a fuc protein -Antibody complex is incubated, b) a lectin selected from the group Ulex-europaeus-agglutinin (UEA), lotus-tetragonolojbus-agglutinin (LTA) and Anguilla-anguilla-agglutinin (AAA) is added, which has appropriate labels to the amount to determine the fuc-protein-antibody complex and therefrom the amount of fucosylated protein in a manner known per se or b) an unlabeled lectin selected from the group mentioned is added and the detection of the fuc-protein-antibody complex by means of labeled anti-lectin antibodies against the lectins mentioned are carried out in a lectin selected from the group Ulex-europa
  • UEA is used as lectin in the sandwich immunoassay according to the invention.
  • Both polyclonal and monoclonal antibodies can be used as capture antibodies on the solid phase.
  • the sandwich immunoassay is carried out in such a way that a lectin labeled with an acceptor selected from UEA, LTA and AAA is added to the immobilized anti-Fuc protein antibody and then with a labeled receptor that binds to the acceptor of the lectin , is incubated.
  • the detection is carried out via the marker on the receptor.
  • Haptens or low molecular weight ligands can be used as acceptors for labeling the lectin; biotin is preferably used. Accordingly, in a preferred embodiment, labeled avidin, streptavidin or their derivatives are used as the receptor. If haptens are used as acceptors, hapten-specific antibodies are used as receptors.
  • enzymes, dyes, radioisotopes, metal colloids, chelators or spin markers can be used as receptor markers.
  • An enzyme such as horseradish peroxidase (POD), alkaline phosphatase, ⁇ -galactosidase, urease or glucose oxidase is preferably used as the receptor marker and the amount of the fuc-protein-antibody complex is detected by a substrate reaction of this enzyme.
  • POD is used for labeling according to the invention and the substrate reaction is carried out by means of a chlorogenic substrate, for example using H0-tetramethylbenzidine.
  • the substrate reactions for the most diverse enzymes can be detected by means of different chromogenic substrates, chemiluminescence or fluorescence.
  • the immunoassay according to the invention is therefore carried out as an enzyme immunoassay.
  • the receptor can also be used with a radioisotope (e.g. 1Z ⁇ : 'J), with a fluorescence marker (e.g. FITC), a metal colloid (e.g. gold), a chelator (e.g. DTTA), with polynucleotides or with a spin marker (e.g. PROXYL) must be marked.
  • the lectin labeled with an acceptor and the receptor as the preformed complex for the antigen-antibody complex add so that a process step is omitted when carrying out the assay variant mentioned.
  • the immunoassay can also be carried out as a direct assay.
  • the lectin according to the invention itself carries the markers mentioned.
  • the implementation as an enzyme immunoassay is preferred.
  • unlabeled lectin is used in the assay according to the invention and the detection is carried out using labeled anti-lectin antibodies.
  • labeled anti-lectin antibodies Both polyclonal and monoclonal antibodies against UEA, LTA or AAA or their fragments can be used.
  • the implementation as an enzyme immunoassay is preferred.
  • a biological sample is to be understood as any sample of a human or animal body fluid that can contain fucosylated proteins.
  • the blood can be plasma, serum, urine, tissue fluid, lymph, gastric juice, ascites or saliva.
  • fucosylated proteins such as, for example, .alpha.-fetoprotein, fucosylated, genetically engineered or pharmaceutical proteins or adhesion proteins (for example CD22) obtained from biological material can be determined.
  • the assay according to the invention is particularly suitable for the differential diagnosis of liver diseases and that the determination of the amount of fucosylated ⁇ -fetoprotein (AFP) in the serum enables a reliable statement to be made about any hepatocellular carcinoma (HCC) present, since the Fucose-specific AFP content in HCC patients is significantly increased (see Fig. 1).
  • HCC hepatocellular carcinoma
  • the AFP assay according to the invention contains polyclonal antibodies or fragments thereof from mouse, rabbit or chicken as capture antibodies on the solid phase.
  • Biotinylated UEA is added to the antigen-antibody complex and the detection of the Fuc-protein-antibody complex is carried out with an avidin, modified avidin or streptavidin-POD conjugate.
  • the technical solution according to the invention thus opens up the possibility of determining the total AFP content, for example by means of a conventional enzyme immunoassay, in the event of suspected liver tumor diseases and, in parallel, determining the content of fucosylated AFP (for example on a microtest plate).
  • the structure of these two assays can, for example, be as shown below: Total AFP Assay:
  • Immobilized anti-AFP antibody or antibody fragment of species 1 e.g. mouse, rabbit, chicken
  • Enzyme-labeled anti-AFP antibody or antibody fragment of species 2 e.g. mouse, rabbit, chicken.
  • Enzyme e.g. Horseradish peroxidase (POD) or alkaline phosphatase (AP)
  • Immobilized anti-AFP antibody or antibody fragment of species 1 e.g. mouse, rabbit, chicken
  • lectin selected from UEA, LTA or AAA (e.g. biotinylated UEA)
  • the invention also relates to the associated reagent sets, as set out in the claims. Examples:
  • Rabbits are immunized with high purity umbilical cord blood human AFP. After coagulation, the antiserum is separated from the blood of the immunized animals by centrifugation.
  • the serum is applied to an AFP-Sepharose. Unbound serum protein is washed out with TRIS-buffered saline (TBS).
  • TRIS-buffered saline TRIS-buffered saline
  • the anti-AFP-IgG is then eluted with a 0.2M glycine-HC1 buffer pH 2.5, neutralized with IM Tris-HCl pH 7.5, concentrated with Centricon 30 kD cartridges and against 50 mM acetate buffer pH 4.0; 0.5M NaCl, 0.02% NaN buffered.
  • the wells of a microtest plate are coated with anti-human AFP-IgG (F (ab) 2 fragment, rabbit) by adsorption from a solution of 10 ⁇ g F (ab) 2 / ml in bicarbonate buffer pH 8.5-9 , 5 occupied.
  • Non-specific protein adsorption is reduced by blocking the plastic surface with an inert protein solution (e.g. bovine serum albumin in phosphate-buffered physiological saline (PBS)).
  • PBS phosphate-buffered physiological saline
  • Either a standard dilution series of human AFP (concentration range 2 to 300 ng / ml) or a centrifuged, 1: 5 to 1:10 diluted sample of patient serum is placed in the wells coated in this way.
  • Unbound material is washed out, then incubated with an appropriate dilution of anti-human AFP-IgG-POD conjugate. Unbound material is washed out, then the substrate reaction is started with H ⁇ O ⁇ tetramethylbenzidine and stopped after a defined time with sulfuric acid. The absorption is measured at 450 nm. The AFP content in the patient's serum is determined using the standard dilution series.
  • the wells of a microtest plate are coated with anti-human AFP-IgG (F (ab) 2 fragment, rabbit), as prepared in example la, by adsorption from a solution.
  • F (ab) 2 fragment, rabbit anti-human AFP-IgG
  • Fig. 1 Content of fucosylated AFP in patient serum samples control - healthy volunteers, SLE - systemic lupus erythromateus, AIH - autoimmune hepatitis, PBC - polynuclear cheap cirrhosis, HCC - hepatocellular carcinoma
  • Fig. 2 Comparison of the total and fucosylated AFP content in different HCC patient sera

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un dosage immunologique en sandwich pour déterminer la quantité de protéine fucosylée (protéine fus) dans un échantillon biologique, ainsi que des ensembles de réactifs correspondants. Un mode préféré de réalisation de l'invention concerne un dosage immunologique permettant de déterminer une foetoprotéine α-fucosylée (AFP) qui est importante pour la détection précoce du carcinome hépatocellulaire (HCC), une forme commune de cancer. Ce dosage immunologique est caractérisé en ce qu'il met en oeuvre une lectine choisie dans le groupe composé de l'agglutinine Ulex-europaeus (UEA), de l'agglutinine Lotus-tetragonolobus (LTA) et de l'agglutine Anguilla-anguilla (AAA).
PCT/EP1999/000639 1998-02-02 1999-02-01 Dosage immunologique et trousse d'essai pour la determination d'une proteine fucosylee dans un echantillon biologique WO1999039209A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2000529612A JP2002502037A (ja) 1998-02-02 1999-02-01 生体試料におけるフコシル化タンパク質の測定のためのイムノアッセイおよび試験キット
EP99904839A EP1053476A1 (fr) 1998-02-02 1999-02-01 Dosage immunologique et trousse d'essai pour la determination d'une proteine fucosylee dans un echantillon biologique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19806185.4 1998-02-02
DE1998106185 DE19806185C2 (de) 1998-02-02 1998-02-02 Immunoassay und Testkit zur Bestimmung von fucosyliertem Protein in einer biologischen Probe

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WO1999039209A1 true WO1999039209A1 (fr) 1999-08-05

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JP (1) JP2002502037A (fr)
DE (1) DE19806185C2 (fr)
WO (1) WO1999039209A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001012212A1 (fr) * 1999-08-13 2001-02-22 The Brigham And Women's Hospital, Inc. Inhibiteurs de la voie du complement a lectine (lpc) et utilisation de ceux-ci
US7273925B1 (en) 1998-12-15 2007-09-25 Brigham And Women's Hospital, Inc. Methods and products for regulating lectin complement pathway associated complement activation
WO2008031288A1 (fr) * 2006-09-13 2008-03-20 Beijing Hotgen Biotech Co., Ltd Colonne centrifuge préremplie servant à dépister un variant d'alpha-fétoprotéine spécifique de l'hépatocarcinome, et trousse d'analyse contenant ladite colonne
US8524453B2 (en) 2006-02-10 2013-09-03 The Brigham And Woman's Hospital, Inc. Lectin complement pathway assays and related compositions and methods
CN107973855A (zh) * 2016-10-24 2018-05-01 希森美康株式会社 与糖肽反应的单克隆抗体及其用途
US20210033627A1 (en) * 2005-05-05 2021-02-04 Drexel University Diagnosis of Liver Pathology Through Assessment of Protein Glycosylation

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9110078B2 (en) 2008-04-04 2015-08-18 Drexel University Diagnosis of liver pathology through assessment of anti-gal IgG glycosylation
FR2980271B1 (fr) * 2011-09-16 2013-10-11 Cisbio Bioassays Procede de determination de la glycosylation d'un anticorps
CN104678103A (zh) * 2014-08-05 2015-06-03 首都医科大学附属北京佑安医院 检测血清糖蛋白岩藻糖指数的化学发光蛋白芯片、试剂盒及检测方法
US20200088737A1 (en) * 2016-01-27 2020-03-19 J-Oil Mills, Inc. Method for Detecting Glycoprotein
JP6935184B2 (ja) * 2016-05-31 2021-09-15 シスメックス株式会社 糖ペプチドと反応するモノクローナル抗体およびその用途

Citations (4)

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Publication number Priority date Publication date Assignee Title
EP0157427A2 (fr) * 1984-04-06 1985-10-09 Otsuka Pharmaceutical Co., Ltd. Procédé de préparation d'un antigène apparenté à la liaison glycosidique
JPS61292062A (ja) * 1985-06-06 1986-12-22 Hidematsu Hirai α−フェトプロテインの分画検出試薬キットおよび分画検出方法
WO1990005304A1 (fr) * 1988-11-10 1990-05-17 Oerntoft Torben Falck Analyses structurelles et enzymatiques sur des glycocomposes, leur emploi dans le diagnostic du cancer, kits utilises dans les analyses et dispositif de prelevement d'un echantillon de secretion via un orifice du corps
WO1997031107A2 (fr) * 1996-02-20 1997-08-28 Coles John G Apoptose induite par des lectines de serum humain et procede de detection d'apoptose

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EP0399464A3 (fr) * 1989-05-24 1992-03-25 Eiji Ishikawa Méthode d'essai par une substance avec une chaíne de sucre spécifique

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0157427A2 (fr) * 1984-04-06 1985-10-09 Otsuka Pharmaceutical Co., Ltd. Procédé de préparation d'un antigène apparenté à la liaison glycosidique
JPS61292062A (ja) * 1985-06-06 1986-12-22 Hidematsu Hirai α−フェトプロテインの分画検出試薬キットおよび分画検出方法
WO1990005304A1 (fr) * 1988-11-10 1990-05-17 Oerntoft Torben Falck Analyses structurelles et enzymatiques sur des glycocomposes, leur emploi dans le diagnostic du cancer, kits utilises dans les analyses et dispositif de prelevement d'un echantillon de secretion via un orifice du corps
WO1997031107A2 (fr) * 1996-02-20 1997-08-28 Coles John G Apoptose induite par des lectines de serum humain et procede de detection d'apoptose

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BALDUS, STEPHAN E. ET AL: "Characterization of the binding specificity of Anguilla anguilla agglutinin (AAA) in comparison to Ulex europaeus agglutinin I (UEA-I)", GLYCOCONJUGATE J. (1996), 13(4), 585-590 CODEN: GLJOEW;ISSN: 0282-0080, XP002105687 *
DATABASE WPI Section Ch Week 8705, Derwent World Patents Index; Class A96, AN 87-034232, XP002105688 *
THOMPSON, STEPHEN ET AL: "A multiwell lectin -binding assay using Lotus tetragonolobus for measuring different glycosylated forms of haptoglobin", CLIN. CHIM. ACTA (1989), 180(3), 277-84 CODEN: CCATAR;ISSN: 0009-8981, XP002105686 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7273925B1 (en) 1998-12-15 2007-09-25 Brigham And Women's Hospital, Inc. Methods and products for regulating lectin complement pathway associated complement activation
WO2001012212A1 (fr) * 1999-08-13 2001-02-22 The Brigham And Women's Hospital, Inc. Inhibiteurs de la voie du complement a lectine (lpc) et utilisation de ceux-ci
US20210033627A1 (en) * 2005-05-05 2021-02-04 Drexel University Diagnosis of Liver Pathology Through Assessment of Protein Glycosylation
US8524453B2 (en) 2006-02-10 2013-09-03 The Brigham And Woman's Hospital, Inc. Lectin complement pathway assays and related compositions and methods
WO2008031288A1 (fr) * 2006-09-13 2008-03-20 Beijing Hotgen Biotech Co., Ltd Colonne centrifuge préremplie servant à dépister un variant d'alpha-fétoprotéine spécifique de l'hépatocarcinome, et trousse d'analyse contenant ladite colonne
CN107973855A (zh) * 2016-10-24 2018-05-01 希森美康株式会社 与糖肽反应的单克隆抗体及其用途

Also Published As

Publication number Publication date
DE19806185C2 (de) 1999-11-18
DE19806185A1 (de) 1999-08-19
EP1053476A1 (fr) 2000-11-22
JP2002502037A (ja) 2002-01-22

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