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WO1995000847A1 - Dosage immunologique - Google Patents

Dosage immunologique Download PDF

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Publication number
WO1995000847A1
WO1995000847A1 PCT/GB1994/001314 GB9401314W WO9500847A1 WO 1995000847 A1 WO1995000847 A1 WO 1995000847A1 GB 9401314 W GB9401314 W GB 9401314W WO 9500847 A1 WO9500847 A1 WO 9500847A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
complex
formation
antibody
protein complex
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/GB1994/001314
Other languages
English (en)
Inventor
Ross Wentworth Stephens
Lars ÖRNING
Kjell Sakariassen
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.)
Hafslund Nycomed AS
Original Assignee
Hafslund Nycomed AS
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 Hafslund Nycomed AS filed Critical Hafslund Nycomed AS
Priority to AU69754/94A priority Critical patent/AU6975494A/en
Publication of WO1995000847A1 publication Critical patent/WO1995000847A1/fr
Anticipated expiration legal-status Critical
Ceased 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
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G01N2333/96441—Serine endopeptidases (3.4.21) with definite EC number
    • G01N2333/96447—Factor VII (3.4.21.21)

Definitions

  • the present invention relates to a method of assaying the formation of multi-protein complexes (such as the FVII/TF complex) , optionally in the presence of test substances, such as peptides.
  • multi-protein complexes such as the FVII/TF complex
  • Antibodies are frequently used by those skilled in protein chemistry for the isolation, purification and/or identification of particular proteins of interest. In this field the availability of monoclonal antibodies has led to great improvements in all techniques relying on the formation of a specific antibody-antigen complex. Monoclonal antibody production (for example by fusing a single antibody-producing cell with a tumour cell to form a stable hybridoma) allows the manufacture of multiple copies of antibodies having identical specificity.
  • assay methods have been developed in which specific antibodies selectively bind the protein of interest.
  • One such well-known assay method is the "solid phase immunoassay" in which the antibody to the protein of interest is immobilised on a polymeric support, for example polyvinylchloride. The antigen-containing test substance is then applied, and any excess antigen is washed off after formation of the antibody-antigen complex. A second antibody (specific for a different site on the antigen) is then added. This second antibody is labelled, for example with a radioactive or fluorescent label, and the amount of label bound is determined.
  • the assay is known as an "enzyme-linked immunosorbent assay" or ELISA.
  • the amount of coloured or fluorescent product formed is a measure of the amount of antigen bound. This particular assay is extremely sensitive and can detect antigen in amounts of less than a nanogram due to the amplifying effect of the enzymic label.
  • the present invention provides an assay for the formation of multi-protein complexes. Additionally, the present invention provides an assay to determine the ability of the test substrates such as peptides or other additives to affect the formation of a multi-protein complex.
  • the assay according to the invention comprises the following steps:
  • step (iv) adding a labelled second antibody specific to a protein added in step (iv) ;
  • washing steps are advantageously performed after each of the stages listed above, to remove any excess reagent present.
  • Water or, more preferably, buffer may be used as the washing medium.
  • the assay of the invention is carried out at a pH in the range of from 7.0 to 7.5, preferably from 7.2 to 7.3.
  • the assay of the present invention can be used to determine whether or to what degree a naturally produced multi-protein complex is formed by an individual. In this way any malfunction in formation of a multi-protein complex, for example due to a genetic disorder or physiological disturbance, can be ascertained. Measurement of the amount of label thus correlates directly to the ability of that patient to form the multi-protein complex.
  • the invention provides an assay as hereinbefore described, in which the said remaining protein added in step (iv) is contained in a body fluid of a patient whereby formation of the labelled multi-protein complex indicates the presence in the body fluid of the said remaining protein in a form capable of forming said complex.
  • test substances of interest are investigated by use of the assay described above.
  • the amount of multi-protein complex formed indicates whether and to what degree the test substance affects the formation of the multi-protein complex.
  • the invention provides an assay as hereinbefore described, in which the test substance added in step (iii) is a substance which may affect formation of said multi-protein complex whereby the ability of the test substance to affect complex formation can be assessed.
  • the first antibody is immobilised on a solid support.
  • Suitable solid supports include beads, tubes, plates, dishes, trays, columns etc. Any free protein binding sites remaining on the support should advantageously be blocked with a blocking protein prior to commencement of the assay.
  • Suitable blocking proteins would be well-known to those skilled in the art and should be selected for their non-involvement with the multi-protein complex under test. Any excess blocking protein should be removed by washing.
  • multi-protein complex is meant an association or combination of at least two different independent proteins which forms spontaneously under suitable conditions. Formation of the multi-protein complex may be reversible.
  • examples of a multi-enzyme complex includes enzyme-substrate complexes, enzyme-inhibitor complexes or a complex of two or more proteins in a functional (e.g. enzymatic) complex, for example on a cell membrane.
  • a multi-protein complex occurs in the extrinsic pathway of the blood clotting cascade, where Factor VII or Vila (FVII or FVIIa) combines with tissue factor (TF) .
  • the FVIIa/TF complex is responsible for catalysing the conversion of Factor X to it's active form in the next stage of the cascade.
  • monoclonal antibodies in the assay of the present invention are, of course, greatly preferred. Either or both of the first and second antibodies may be monoclonal. Monoclonal antibodies to a wide variety of proteins are commercially available and the manufacture of monoclonal antibodies to a particular protein is within the ability of the skilled man. In order to determine the amount of second antibody bound by the assay, that antibody must be labelled. Excess second antibody is removed by washing prior to measurement. The second antibody may be labelled in any known manner and several suitable labels are available.
  • the second antibody may be labelled with a chromophore or fluorophore such as dansylchloride or with a radioactive atom such as 125 I .
  • Other labels include enzymes, such as an alkaline phosphatase or peroxidase which catalyse the formation of detectable products, and colloidal metals such as gold.
  • the present invention also provides the use of an antibody to a component of a multi-protein complex in the assay described above.
  • the present invention provides a kit for determining the effect, ie enhancement or inhibition, of a test substance on the formation of a multi-protein complex, said kit comprising means for forming a, preferably immobilised, first antibody-first protein conjugate and separately thereto a second protein of the multi-protein complex, and a labelled second antibody specific therefor.
  • the kit may also include buffer or water for washing between each step of the assay procedure.
  • the antibodies provided in the kit are monoclonal .
  • the kit according to the invention may also comprise the substrate for the particular enzyme used.
  • a reagent to halt the reaction catalyzed by the enzyme-label may additionally be provided.
  • the present invention also provides a method for the in vitro diagnosis of abnormal formation of multi-protein complexes, said method comprising assaying a body fluid (or extract or diluent thereof) with the assay described above.
  • Body fluids suitable for the assay procedure include blood, serum, urine, saliva, plasma etc.
  • Figure 1 is a schematic diagram of an assay complex according to the invention, involving the multi-protein complex of FVII/TF.
  • Figure 2 indicates the binding of FVII to immobilised rTF (recombinant tissue factor) at different coating concentrations as a function of FVII concentration.
  • Figure 3 illustrates the requirement for calcium ions in formation of the FVII/TF multi-protein complex (filled diamonds) . Open squares represent controls without tissue factor.
  • Figure 4 illustrates how formation of the FVII/TF multi- protein complex is affected by addition of increasing concentrations of a monoclonal antibody which binds to FVII in competition with TF.
  • Figure 5 illustrates how formation of the FVII/TF multi-protein complex is affected by addition of various synthetic peptides.
  • Figure 6 shows FVII from normal plasma at various dilutions binding to immobilised rTF.
  • Example 1 Assay method with the multi-protein complex FVII/TF
  • 96 well immunoassay plates (Nunc Maxisorp cat# 442404) were coated with a monoclonal antibody to human tissue factor (American Diagnostica cat# 4503) as follows: a solution of the antibody at 5 ⁇ g IgG/ml was made up in 0.05 M Tris. HC1 pH 9.5 and 100 ⁇ l was pipetted into each well. The wells were then covered with a plastic sealer, and the plates were incubated for 15 hours at 37°C. They were then washed twice with 250 ⁇ l/well of 10 mM Tris.HCl pH 7.3 containing 5 mM CaCl 2 and 0.05% Tween 20 (hereafter referred to as TCCT) .
  • TCCT Tween 20
  • the non-specific binding of the wells was blocked by allowing 150 ⁇ l/well of 1 mg/ml bovine serum albumin (BSA) in TCCT to stand in the wells for 1 hour at 23°.
  • BSA bovine serum albumin
  • TCCT/BSA TCCT/BSA
  • FVII solution 0.1 ⁇ g/ml in TCCT/BSA
  • TCCT/BSA TCCT/BSA
  • Example 2 Effect of synthetic peptides on formation of FVII/TF multi-protein complex.
  • FVII peptides tested the most inhibition of FVII binding to rTF was obtained with cyclic peptides representing the ring structures present in the FVII growth factor domains. Some activity was also present in a linear peptide of residues 136-146 from a region close to the site of cleavage which activates FVII.
  • Example 3 Assay of human plasma for ability to form the multi-protein complex FVII/TF
  • the binding assay of Example 1 was used to test the binding ability of FVII from human plasma.
  • the assay was performed using normal plasma, plasma immunodepleted of FVII (Diagnostica Stago) and plasma from a patient having a homozygous mutation at residue 100 (glutamine to arginine) in the second growth factor (GF) domain of FVII.
  • the plasma samples were diluted in TCCT buffer containing 50 ⁇ m p-nitrophenyl guanidinobenzoate to inhibit all serine proteases of the clotting pathway.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Dosage utilisé pour préparer des complexes multiprotéiques, comprenant les étapes suivantes: (i) on fait réagir une première protéine d'un complexe multiprotéique avec un premier anticorps immobiisé spécifique contre ce dernier qui n'interfère pas avec la formation du complexe; (ii) on ajoute, de manière facultative, d'autres protéines qui font partie du complexe multiprotéique; (iii) on ajoute facultativement une substance de test; (iv) on ajoute la (ou les) protéine(s) restante(s) nécessaire(s) pour la formation du complexe multiprotéique; (v) on ajoute un deuxième anticorps étiqueté spécifique contre une protéine ajoutée dans l'étape (iv); et (vi) on détecte et on détermine, facultativement, la quantité du deuxième anticorps immobilisé pour obtenir une indication de la formation du complexe multiprotéique. On peut utiliser ce dosage pour déterminer s'il se forme chez un individu un complexe multiprotéique produit naturellement ou bien dans quelle mesure un complexe de ce type se forme. De cette manière on peut constater n'importe quel défaut se produisant dans la formation d'un complexe multiprotéique, par exemple à cause d'un dérèglement génétique ou d'une perturbation physiologique.
PCT/GB1994/001314 1993-06-18 1994-06-17 Dosage immunologique Ceased WO1995000847A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU69754/94A AU6975494A (en) 1993-06-18 1994-06-17 Immunoassay

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9312638.1 1993-06-18
GB939312638A GB9312638D0 (en) 1993-06-18 1993-06-18 Assay

Publications (1)

Publication Number Publication Date
WO1995000847A1 true WO1995000847A1 (fr) 1995-01-05

Family

ID=10737408

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1994/001314 Ceased WO1995000847A1 (fr) 1993-06-18 1994-06-17 Dosage immunologique

Country Status (4)

Country Link
AU (1) AU6975494A (fr)
GB (1) GB9312638D0 (fr)
WO (1) WO1995000847A1 (fr)
ZA (1) ZA944336B (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996018654A1 (fr) * 1994-12-15 1996-06-20 Nycomed Imaging As Fragment 82-128 du facteur vii et son utilisation pour le traitement des troubles de la coagulation sanguine
EP2251689A1 (fr) * 2006-03-31 2010-11-17 Karima Ferrani-Kile Procédés et compositions pour la préparation et l'utilisation de complexes multiprotéines biologiquement actifs

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0048989A2 (fr) * 1980-09-30 1982-04-07 Cornell Research Foundation, Inc. Procédé pour la détermination des complexes inhibiteurs d'enzymes
EP0446797A2 (fr) * 1990-03-13 1991-09-18 BEHRINGWERKE Aktiengesellschaft Peptides synthétiques comportant des séquences du facteur VIIa et leur utilisation
EP0450086A1 (fr) * 1989-10-02 1991-10-09 Teijin Limited Kit servant a l'immunoanalyse de complexes d'activateur de plasminogene tissulaire humain/inhibiteur d'activateur de plasminogene humain et procede d'immunoanalyse

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0048989A2 (fr) * 1980-09-30 1982-04-07 Cornell Research Foundation, Inc. Procédé pour la détermination des complexes inhibiteurs d'enzymes
EP0450086A1 (fr) * 1989-10-02 1991-10-09 Teijin Limited Kit servant a l'immunoanalyse de complexes d'activateur de plasminogene tissulaire humain/inhibiteur d'activateur de plasminogene humain et procede d'immunoanalyse
EP0446797A2 (fr) * 1990-03-13 1991-09-18 BEHRINGWERKE Aktiengesellschaft Peptides synthétiques comportant des séquences du facteur VIIa et leur utilisation

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996018654A1 (fr) * 1994-12-15 1996-06-20 Nycomed Imaging As Fragment 82-128 du facteur vii et son utilisation pour le traitement des troubles de la coagulation sanguine
US5948759A (en) * 1994-12-15 1999-09-07 Nycomed Imaging As Factor VII fragment 82-128 and its use in blood-clotting disorders
EP2251689A1 (fr) * 2006-03-31 2010-11-17 Karima Ferrani-Kile Procédés et compositions pour la préparation et l'utilisation de complexes multiprotéines biologiquement actifs
AU2007233092B2 (en) * 2006-03-31 2014-03-20 Karima Ferrani-Kile Methods and compositions for obtaining and using biologically active multi-protein complexes

Also Published As

Publication number Publication date
AU6975494A (en) 1995-01-17
GB9312638D0 (en) 1993-08-04
ZA944336B (en) 1995-02-27

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