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WO1993015406A1 - Procedes et dispositif de dosage de polysaccharides sulfates - Google Patents

Procedes et dispositif de dosage de polysaccharides sulfates Download PDF

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Publication number
WO1993015406A1
WO1993015406A1 PCT/GB1993/000197 GB9300197W WO9315406A1 WO 1993015406 A1 WO1993015406 A1 WO 1993015406A1 GB 9300197 W GB9300197 W GB 9300197W WO 9315406 A1 WO9315406 A1 WO 9315406A1
Authority
WO
WIPO (PCT)
Prior art keywords
polymer
polysaccharide
assay
reporter group
binding
Prior art date
Application number
PCT/GB1993/000197
Other languages
English (en)
Inventor
Anthony Edward George Cass
Kalvinder Sohanpal
Original Assignee
Imperial College Of Science, Technology & Medecine
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 Imperial College Of Science, Technology & Medecine filed Critical Imperial College Of Science, Technology & Medecine
Publication of WO1993015406A1 publication Critical patent/WO1993015406A1/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
    • G01N33/86—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
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, Konjac gum, Locust bean gum or Guar gum
    • G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides

Definitions

  • the present invention is concerned with detection techniques, apparatus and probes for the measurement of sulphated polysaccharides and especially heparin.
  • Heparin is a sulphated polysaccharide that is a potentiator of the anticoagulent activity of anti thrombin III, a naturally occurring anticoagulent present in the blood. Heparin is widely used in surgery to prevent blood coagulation during major operative procedures; the monitoring of its levels during the operation and appropriate adjustment of dose prevents premature coagulation during surgery.
  • Heparin is a polyanionic material and binds to many proteins such as antithrombin III, lipoprotein lipase, histidine rich glycoprotein, la inin and collagen. Many of these interactions are responsible for heparin's physiological functions. It will also bind to antiheparin antibodies and heparinase as well as synthetic homo-and hetero-polypeptides.
  • the present invention is based on measuring heparin by the interaction between heparin and a complementary binding polymer (CBP) such as a protein, the interaction between the two molecules causing physiochemical change in the properties of a reporter group attached to the CBP. In the reporter group, binding of heparin to the CBP can alter an optical property such as fluorescence.
  • CBP complementary binding polymer
  • a method of performing an assay on a sample which may or does contain a sulphated polysaccharide of interest which comprises contacting the sample with (i) a complementary binding polymer (CBP) which is labelled with (ii) at least one optically active reporter group and which polymer is capable of forming (iii) a complex with said polysaccharide, said reporter group being sensitive to formation and/or presence of said complex, followed by measuring change in an optical property of the optically- labelled polymer as a result of such contact, and comparing the change with values derived from a control assay for the same polysaccharide, polymer and reporter group and thereby to determine the concentration of the polysaccharide of interest in the sample.
  • CBP complementary binding polymer
  • CBP complementary binding polymer
  • the polymer (i) being labelled with at least one optically active reporter group as specified hereinabove, and capable of forming
  • an insoluble support for the optically labelled, complex-forming binding polymer is more preferably immobilised and may form a component part of heparin concentration detection means, such as an optical probe attached to optical fibre upon which alteration in the optical property, notably quenching of fluorescence, can be monitored.
  • an immobilised support such as a solid, unreactive surface or polymer
  • that support may comprise or be part of an electrode, probe or optical waveguide.
  • the binding of heparin to a synthetic polycationic peptide or other polymer is measured by the change in the fluorescence intensity of a r fluorophore' group attached to the polymer.
  • the polymer is either poly(L- ornithine), poly(L-lysine) or poly(ethylenei ine) although other examples are possible.
  • the fluorescence reporter group may be, for example either fluorescein or tetramethylrhodamine although other types of fluorescent group might behave similarly.
  • the fluorescent group is attached to the CBP through the letter's amino groups (in the case of polypeptide) although other chemical linkages may be employed.
  • the ratio of fluorescent group to monomer unit can be varied to optimise the response range of the assay. In the embodiments described herein, quenching of fluorescence intensity is measured to monitor the reaction.
  • the reaction between the CBP and heparin occurs on a solid surface which also acts to guide in the excitation light from a source and to carry the emitted light to a detector.
  • a solid surface which also acts to guide in the excitation light from a source and to carry the emitted light to a detector.
  • the reaction is carried out on a solid surface attached to the optical system and the fluorescence is subsequently measured or those where the solid surface is placed in a fluorescence spectrometer or those where the complex formation between heparin and the labelled CBP is carried out in solution and the fluorescence properties of the solution are subsequently measured.
  • the CBP as defined herein, have polycationic domains which are capable of complex formation with the anionic domains of the sulphated polysaccharide.
  • the most preferred polysaccharide is heparin.
  • the polymer selected may have various molecular weights, and the dose-response curve may be dependent on the molecular weight.
  • he CBP is also preferably hydrophilic. It is preferred to select a complementary binding polymer which is bio-compatible and which might be effective and thus suitable for in vivo use.
  • Fluorescence as the optical property is preferred, and the fluorescent label (fluorophore) preferably exhibits fluorescence in the visible to near-infrared wavelength range.
  • fluorescent label fluorophore
  • the complementary binding polymer should preferably only be chemically reactive in the sense that it can be fluorescently labelled and bind ionically to the polysaccharide, preferably a strongly anionic polysaccharide such as heparin.
  • the fluorescent label selected should still fluoresce when bound to the said polymer.
  • the optical property can increase or decrease in the presence of the polysaccharide of interest, although with heparin it appears quenching of fluorescent activity is sensitive to increase in heparin concentration.
  • the notional skilled worker need only conduct simple laboratory experiments. In short, if a detectable change occurs in the optical property of the optically active reporter group in response to variation in e.g. heparin concentration, then that reporter group and polymer are potentially usable.
  • Means for assaying heparin in accordance with the invention may be supplied in kit form, in which case the kit may additionally comprise a solid surface to which the CBP would bind e.g. polystyrene microtitre plates, polystyrene or polyamide film for external analysis or probe means comprising an immobilised support for binding purposes wherein there is preferably covalent linkage between the immobilised support and the labelled CBP.
  • kit form in which case the kit may additionally comprise a solid surface to which the CBP would bind e.g. polystyrene microtitre plates, polystyrene or polyamide film for external analysis or probe means comprising an immobilised support for binding purposes wherein there is preferably covalent linkage between the immobilised support and the labelled CBP.
  • a light source will be needed for photometric quantitative determinations preferably incorporating means for selecting a small band of wavelength of maximum absorption of the label in use, coupled with means for applying light to the labelled polymer such as a lens, an air gap or an optical fibre. Additionally it may be necessary to incorporate means, such as filter means, to select the wavelength of light emitted from the sample under investigation, means such as an optical fibre to direct emitted light to a photodetector and means such as £C photodiode or photomultiplier tube to detect the intensity of the emitted light from the labelled polymer. Apparatus for such purposes is shown schematically in Figures 2a and 2b .
  • readings of light intensity can be taken via a Perkin Elmer fluorimeter LS 50 coupled with data processing means which store and interpret previously assayed standards - calibration curves which plot, for the CBP - fluorophore in use, intensity of emitted light against concentration of heparin over the therapeutic range.
  • a baseline reading with no heparin present may be first taken and other readings taken which may be specified as a percentage change.
  • Sensitivity and accuracy of the readings can be influenced by selection of (a) the flurophore, (b) the ratio of fluorophore groups to repeating monomer units and (c) the nature of the polymer, e.g. protein, selected as the CBP.
  • Figure 1 shows the molecular structures of some possible polycationic CBPs
  • Figure 2 shows the molecular structures of some 'fluorophores* that can be used to label the polycations shown in Figure 1, or other suitable polycationic molecules,
  • Figure 2a shows one form of optical fibre fluorimeter including movable probe as an embodiment of a heparin concentration detector.
  • Figure 2b is a schematic view of components suitable for conducting the assays on a fixed, immobilised support plate
  • Figures 3a and 3b show fluorescence emission spectra of some the reporter groups of Figure 2 attached to some of the CBPs of Figure 1, whereby Figure 3a shows the fluorescence emission spectra of fluorescein isothiocyanate (FITC) bound to poly-L-ornithine, using an excitation wavelength of 495nm and scanning the emission from 500-550nm.
  • FITC fluorescein isothiocyanate
  • Figure 3b shows the fluorescence emission spectra of tetra ethylrhodamine isothiocyanate bound to poly-L- ornithine as a polymer conjugate using an excitation wavelength of 550mm and scanning the emission from 560- 600mm,
  • Figure 4 shows the quenching effect of heparin upon the fluorescence intensity, (at 520nm of the fluorophore (FITC) attached to different molecular weight polymer of poly-L- lysine.
  • the polymer used in this study was poly-L-ornithine (m.wt 19,900) and the fluorophore was FITC, and
  • Figure 2 shows labelling of binding polymer (4) with fluoroscein isothiocyanate (3) FITC to form fluorescently labelled polymer (5) and labelling of binding polymer (4) with tetramethylrhodamine isothiocyanate (6) TRITC to form the labelled polymer (7) .
  • Figure 2a shows one embodiment of a fibre fluorimeter in which three optical fibres 1 are linked via beam splitter 2.
  • the fibre end 3 shows an expanded view with a binding protein immobilised thereon.
  • Light from a light source 5 passes through excitation filter 4 to the fibre end 3, and emitted light passes through emission filter 6 to a photodetector 7.
  • the end 3 can be a probe for in vivo use.
  • FIG. 2b An alternative arrangement in Figure 2b shows a wave guide 3b with binding protein immobilised on a solid surface thereof.
  • Light from light source 5 causes excitation indicated by arrow 8, the binding protein fluoresces indicated by arrow 9, which is measured by photodetector 7 after passing through emission filter 6.
  • Figure 4 shows the effect of heparin on the fluorescence emission intensity of a fluorescein group bound to poly(L- lysince) of differing molecular weights and illustrates how, by choice
  • the dose-response curve can be adjusted for different concentration ranges.
  • Figure 5 shows the effect of heparin on the fluorescence emission intensity of a fluorescein group bound to poly(ethyleneimine) of differing molecular weights and illustrates how, by choice of the appropriate CBP, the dose- response curve can be adjusted for different concentration ranges.
  • Figure 6 shows the effect of heparin on the fluorescence emission intensity of a tetramethylrhodamine group bound to poly(ethylenei ine) of differing molecular weights and illustrates how, by choice of the appropriate CBP, the dose-response curve can be adjusted for different concentration ranges.
  • Figure 7 shows the effect of heparin on the fluorescence emission intensity of a fluorescein group bound to poly(L-ornithine) of differing degrees of labelling and illustrates how, by choice of the appropriate labelling conditions, the dose-response curve can be adjusted for different concentration ranges.
  • Figure 8 shows the effect of heparin on the fluorescence emission intensity of a tetramethylrhodamine group bound to poly(L-ornithine) of differing degrees of labelling and illustrates how, by choice of the appropriate labelling conditions, the dose-response curve can be adjusted for different concentration ranges.
  • the data from any of Figures 3a to 8 provide a quantitative relationship between fluorescence alteration and heparin level.
  • the data can be stored as a known, control assay in e.g. a computerised database on suitable hardware and (software) including a monitor or other display for reading off the heparin level, using parameters derived from the given fluorescent label in use. Less preferably evaluation and determination of the heparin assay could be effected manually, by using the appropriate graph as a calibrating guide.
  • the dose-response curve for heparin can be tuned over a wide range by appropriate adjustment of the following properties:
  • CBP include those designed to minimise interference from intrinsic blood constituents, to improve the stability of the material, to aid the attachment to a solid surface.
  • the present invention has advantages over the currently used systems in that the detection apparatus and method can be used for in vivo testing of heparin level in a suitably miniaturised form e.g. by using a probe comprising immobilised support, and a continuous "real time" display of actual heparin concentration.
  • the apparatus can be relatively simple in mechanical and optical construction, and may not require use by highly skilled operatives.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
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  • Food Science & Technology (AREA)
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  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

Procédé de dosage d'un échantillon afin d'effectuer des mesures de polysaccharide sulfaté et comprenant la mise en contact dudit échantillon avec (i) un polymère de liaison complémentaire capable de se fixer audit polysaccharide, ledit polymère de liaison étant marqué par (ii) au moins un groupe rapporteur optiquement actif, ledit polymère (i) étant capable de constituer (iii) un complexe avec ledit polysaccharide, le groupe rapporteur étant sensible à la formation et/ou à la pression dudit complexe; la mesure de la modification d'une propriété optique du polymère marqué optiquement consécutivement à ladite mise en contact; la comparaison de la modification de ladite propriété optique avec des valeurs calculées à partir d'un dosage témoin effectué sur ledit polysaccharide, ledit polymère de liaison et ledit groupe rapporteur; l'utilisation de la comparaison des valeurs entre les dosages d'échantillonnage et témoin, de façon à déterminer la concentration de polysaccharide dans ledit échantillon. Le polysaccharide préféré est l'héparine, le polymère de liaison complémentaire est, de préférence, un polypeptide polycationique et la propriété optique est, de préférence, la fluorescence. Les dosages effectués au moyen dudit procédé peuvent être plus rapides que ceux correspondant à l'état actuel de la technique et peuvent même être appropriés à une utilisation in vivo.
PCT/GB1993/000197 1992-01-30 1993-01-29 Procedes et dispositif de dosage de polysaccharides sulfates WO1993015406A1 (fr)

Applications Claiming Priority (2)

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GB929202019A GB9202019D0 (en) 1992-01-30 1992-01-30 Methods and apparatus for assay of sulphated polysaccharides
GB9202019.7 1992-01-30

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5869517A (en) * 1994-07-06 1999-02-09 Basf Aktiengesellschaft 2- (dihydro)pyrazol-3'-yloxymethylene!anilides, their preparation and their use
WO2000042222A2 (fr) * 1999-01-15 2000-07-20 Gene Logic Inc. Reactif d'hybridation d'acide nucleique immobilise et procede associe
US6869789B2 (en) 2000-03-08 2005-03-22 Massachusetts Institute Of Technology Heparinase III and uses thereof
US7056504B1 (en) 1998-08-27 2006-06-06 Massachusetts Institute Of Technology Rationally designed heparinases derived from heparinase I and II
US7083937B2 (en) 2000-09-12 2006-08-01 Massachusetts Institute Of Technology Methods and products related to the analysis of polysaccarides
US7110889B2 (en) 1999-04-23 2006-09-19 Massachusetts Institute Of Technology Method for identifying or characterizing properties of polymeric units
US7560106B2 (en) 1998-08-27 2009-07-14 Massachusetts Institute Of Technology Rationally designed heparinases derived from heparinase I and II and methods of sequencing therewith
US7709461B2 (en) 2000-10-18 2010-05-04 Massachusetts Institute Of Technology Methods and products related to pulmonary delivery of polysaccharides
US8119357B2 (en) 2003-12-18 2012-02-21 Procognia, Ltd. Method for analyzing a glycomolecule

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0281128A1 (fr) * 1987-03-04 1988-09-07 Slovenska Akademia Vied Procédé et préparation pour séparer l'héparnic du sang in vitro
EP0362077A1 (fr) * 1988-09-29 1990-04-04 Elf Sanofi Procédé de dosage de polyosides anioniques sulfates, et coffret pour la mise en oeuvre de ce procédé
FR2640385A1 (fr) * 1988-12-08 1990-06-15 Serbio Nouveau procede de dosage de l'heparine et son utilisation dans les trousses de dosage
DE4020966A1 (de) * 1990-06-30 1992-01-16 Hoechst Ag Verfahren zum nachweis schwefelhaltiger anionischer polymere sowie test-kit zu dessen durchfuehrung

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0281128A1 (fr) * 1987-03-04 1988-09-07 Slovenska Akademia Vied Procédé et préparation pour séparer l'héparnic du sang in vitro
EP0362077A1 (fr) * 1988-09-29 1990-04-04 Elf Sanofi Procédé de dosage de polyosides anioniques sulfates, et coffret pour la mise en oeuvre de ce procédé
FR2640385A1 (fr) * 1988-12-08 1990-06-15 Serbio Nouveau procede de dosage de l'heparine et son utilisation dans les trousses de dosage
DE4020966A1 (de) * 1990-06-30 1992-01-16 Hoechst Ag Verfahren zum nachweis schwefelhaltiger anionischer polymere sowie test-kit zu dessen durchfuehrung

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 105, no. 17, 27 October 1986, Columbus, Ohio, US; abstract no. 145902s, G. R. JONES ET AL. 'A COMPARISON OF THE STRENGTH OF BINDING OF ANTITROMBIN III, PROTEMINE AND POLY(L-LYSINE) TO HEPARIN SAMPLES OF DIFFERENT ANTICOAGULANT ACTIVITIES.' page 41-42 ; *
PATENT ABSTRACTS OF JAPAN vol. 3, no. 104 (C-57)4 September 1979 *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5869517A (en) * 1994-07-06 1999-02-09 Basf Aktiengesellschaft 2- (dihydro)pyrazol-3'-yloxymethylene!anilides, their preparation and their use
US7560106B2 (en) 1998-08-27 2009-07-14 Massachusetts Institute Of Technology Rationally designed heparinases derived from heparinase I and II and methods of sequencing therewith
US7056504B1 (en) 1998-08-27 2006-06-06 Massachusetts Institute Of Technology Rationally designed heparinases derived from heparinase I and II
WO2000042222A2 (fr) * 1999-01-15 2000-07-20 Gene Logic Inc. Reactif d'hybridation d'acide nucleique immobilise et procede associe
WO2000042222A3 (fr) * 1999-01-15 2000-11-30 Gene Logic Inc Reactif d'hybridation d'acide nucleique immobilise et procede associe
US6312906B1 (en) 1999-01-15 2001-11-06 Imperial College Innovations, Ltd. Immobilized nucleic acid hybridization reagent and method
US7412332B1 (en) 1999-04-23 2008-08-12 Massachusetts Institute Of Technology Method for analyzing polysaccharides
US7110889B2 (en) 1999-04-23 2006-09-19 Massachusetts Institute Of Technology Method for identifying or characterizing properties of polymeric units
US7117100B2 (en) 1999-04-23 2006-10-03 Massachusetts Institute Of Technology Method for the compositional analysis of polymers
US7139666B2 (en) 1999-04-23 2006-11-21 Massachusetts Institute Of Technology Method for identifying or characterizing properties of polymeric units
US7390633B2 (en) 2000-03-08 2008-06-24 Massachusetts Institute Of Technology Methods for preparing low molecular weight heparin with modified heparinase III
US7455986B2 (en) 2000-03-08 2008-11-25 Massachusetts Institute Of Technology, Inc. Heparinase III and methods of specifically cleaving therewith
US6869789B2 (en) 2000-03-08 2005-03-22 Massachusetts Institute Of Technology Heparinase III and uses thereof
US7939292B2 (en) 2000-03-08 2011-05-10 Massachusetts Institute Of Technology Modified heparinase III and methods of sequencing therewith
US7399604B2 (en) 2000-09-12 2008-07-15 Massachusetts Institute Of Technology Methods and products related to evaluating the quality of a polysaccharide
US7083937B2 (en) 2000-09-12 2006-08-01 Massachusetts Institute Of Technology Methods and products related to the analysis of polysaccarides
US7585642B2 (en) 2000-09-12 2009-09-08 Massachusetts Institute Of Technology Methods for evaluating the quality of a heparin sample
US7687479B2 (en) 2000-09-12 2010-03-30 Massachusetts Institute Of Technology Methods and producing low molecular weight heparin
US7709461B2 (en) 2000-10-18 2010-05-04 Massachusetts Institute Of Technology Methods and products related to pulmonary delivery of polysaccharides
US8119357B2 (en) 2003-12-18 2012-02-21 Procognia, Ltd. Method for analyzing a glycomolecule

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AU3456493A (en) 1993-09-01
GB9202019D0 (en) 1992-03-18

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