[go: up one dir, main page]

WO1993002356A1 - Analyse de glucides et necessaire d'analyse - Google Patents

Analyse de glucides et necessaire d'analyse Download PDF

Info

Publication number
WO1993002356A1
WO1993002356A1 PCT/US1991/004555 US9104555W WO9302356A1 WO 1993002356 A1 WO1993002356 A1 WO 1993002356A1 US 9104555 W US9104555 W US 9104555W WO 9302356 A1 WO9302356 A1 WO 9302356A1
Authority
WO
WIPO (PCT)
Prior art keywords
gel
kit
substances
carbohydrate
concentration
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/US1991/004555
Other languages
English (en)
Inventor
Peter Jackson
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.)
Astroscan Ltd
Glyko Inc
Original Assignee
Astroscan Ltd
Glyko 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 Astroscan Ltd, Glyko Inc filed Critical Astroscan Ltd
Priority to PCT/US1991/004555 priority Critical patent/WO1993002356A1/fr
Priority to JP3513054A priority patent/JPH06504363A/ja
Priority to EP91913717A priority patent/EP0595806A1/fr
Publication of WO1993002356A1 publication Critical patent/WO1993002356A1/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
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • 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

Definitions

  • This invention concerns analysis of carbohydrates.
  • the present invention concerns a development of such techniques, and is based on the use of alternative materials as the fluorescent labelling reagents.
  • a method of separating or distinguishing carbohydrate substances comprising labelling carbohydrate substances with labelling reagent containing a hydrazide group to produce flourescently labelled substances, applying the labelled substances to an electrophoretic gel, and running the gel to cause differential migration of different substances.
  • kits for separating or distinguishing carbohydrate substances comprising a labelling reagent containting a hydrozide group capable of producing flourescently labelled substances, an electrophoretic gel, and a carbohydrate standard to compare the carbohydrate(s) substance to be tested.
  • the kit may additionally include a charge couples device.
  • the hydrazide group of the labelling reagent reacts readily with carbohydrates containing a reducing end group, producing a derivate which is both charged and fluorescent and which may be well suited to electrophoretic treatment.
  • the structural formula for the dipotassium salt is shown is Figure 1. Lucifer Yellow CH and its dipotassium salt are available from Aldrich Chemical Company, Inc., Molecular Probes, Inc., Sigma Chemical Company. Also see Stewart.W;Cell.14. 741 (1978), incorporated herein by reference. Modified versions and derivatives of LYCH may give similar results.
  • hydrazide group-containing reagents A number of other hydrazide group-containing reagents are also known, and tests have been carried out with some.
  • hydrazinoacridone- labelled carbohydrates produced blue fluorescent bands after electrophoretic treatment. The bands became bright turquoise after drying.
  • Use of lissamine rhodamine B sulfonyl hydrazide as labelling reagent produced photographable sugar ' derivative bands after being run on an electrophoretic gel.
  • the labelled carbohydrate substance must be fluorescent: the labelling reagent may itself be fluorescent, or may become fluorescent after reaction with the carbohydrate substance.
  • the gel preferably comprises a relatively dense polyacrylamide gel, having a concentration in the range 15% to 60%, preferably 20% to 40%, although in some cases it may be possible or preferable to use gels of lower concentration.
  • the gel may be either of uniform concentration, or in the form of a gradient gel.
  • the gel is preferably cross linked, e.g. with N,N' methylenebisacrylamide (bis).
  • One presently preferred gel comprises a linear polyacrylamide gradient gel having a polyacrylamide concentration (w/v) varying in a continuous gradient from 20% (top) to 40% (bottom).
  • the gel is crosslinked with bis, at a concentration (w/v) varying from 0.53% at the lowest concentration of polyacrylamide to 1.06% at the highest concentration of polyacrylamide.
  • the gel is preferably run using stacking buffer systems (also known as moving boundary electrophoresis, multiphasic zone electrophoresis and other names), using techniques known for working with protein and DNA fragments, e.g. as described in the book "Gel electrophoresis of proteins: a practical approach” edited by B. D. Hames and D. Rickwood, published by IRL Press.
  • stacking buffer systems also known as moving boundary electrophoresis, multiphasic zone electrophoresis and other names
  • Electrophoresis is conveniently carried out using the discontinuous electrophoretic buffer system based on that described in Neville, Jr. D.M., J. Biol. Chem. 246. 6328-6334.
  • the labelled carbohydrate substances when illuminated with light of suitable wavelength, e.g. ultra violet, may be visible with the naked eye in some cases, although better resolution and sensitivity may be obtained by imaging with the light of suitable wavelength, e.g. ultra violet.
  • suitable wavelength e.g. ultra violet
  • CCD CCD-Coupled Device Code Division Multiple Access
  • LYCH fluoresces strongly with a yellow emission and a high quantum yield.
  • the emission is well suited for detection by a CCD, which has greatest sensitivity and quantum efficiency at the red end of the spectrum and lowest sensitivity and quantum efficiency at the blue end of the spectrum.
  • Use of a CCD also has the advantage of giving readily quantitated results very quickly. Good quantitive results are easily available with a CCD due to its wide linear dynamic range. Further, a CCD can be used to view the gel while it is being run.
  • a cooled 2-D CCD operating in slow scan readout.
  • One example of a suitable CCD system is the CCD 2200 Imaging System produced by Astromed Limited, Cambridge, United Kingdom.
  • the CCD is preferably cooled to as least as low as -25'C, with sensitivity being significantly increased by further cooling down as far as -160'C.
  • Typical operation temperatures are in the range -40 * C to -130'C.
  • the labelling reagent e.g., LYCH may be attached to sites on the carbohydrate substances, after release if necessary from an attached biomolecule.
  • the biomolecule may be modified in known way to enable incorporation of the labelling reagent.
  • a carbohydrate substance may be labelled with LYCH by incubating the substance with LYCH, possibly in the presence of a reducing agent, e.g. sodium ⁇ yanoborohydride.
  • a reducing agent e.g. sodium ⁇ yanoborohydride.
  • the sodium cyanoborohydride is preferably in solution in dimethylsulphoxide (DMSO) .
  • LYCH is commercially available, eg form Molecular Probes Inc., Eugene,
  • the rate of migration of substances undergoing electrophoresis varies with the size (molecular weight) and structure of the substances.
  • the invention may thus be used to obtain information on the size and shape of carbohydrate substances, and by comparing results with those for known standards it may be possible partly or fully to characterize an unknown carbohydrate substance.
  • One use of the invention is in elucidating carbohydrate structures, as described in O88/10422, incorporated herein by reference by cleaving an unknown carbohydrate into smaller fragments by use - of glycosidases and identifying the resulting fragments.
  • the invention is applicable to use on a wide range of carbohydrate structures, including those derived from glycoproteins, proteoglycans, glycolipid ⁇ and glycosphingolipids and other biomolecules.
  • Figure 1 illustrates the structure of LYCH in the form of dipotassium salt
  • Figure 2 is a paragraph of an electrophoretic gel showing various sugars labelled with LYCH.
  • LYCH was obtained as its dipotassium salt from Molecular Probes Inc. A fine suspension (5.21g/l) was made in glacial acetic acid/water (15:85,v/v). Sodium cyanoborohydride (NaCNBH 3 ) was obtained from Aldrich Chemical Co. and uses as a 0.1M solution in di ethylsulphoxide (DMSO) .
  • DMSO di ethylsulphoxide
  • a solution containing 5nmol of each of the following reducing saccharides (all 'D' configuration) was freeze-dried into a icrocentrifuge tube: 6- deoxyglucose, glucose, galactose, N-acetylgalctose, galactosyl alpha 1-4 galactose, lactose, maltose, galactobiose, cellotriose, maltotriose, altotetraose, maltopentaose, maltohexaose and maltoheptaose.
  • To the dried saccharides was added 5ul of the LYCH suspension and 5ul of the sodium cyanoborohydride solution.
  • the resulting solution was mixed well, incubated for 30 minutes at room temperature and freeze-dried at 40 * C for 4h using a centrifugal vacuum evaporator.
  • a suitable volume of a solution (electrophoresis sample buffer) containing 6M-urea in 0.041M Tris base, O.04M boric acid pH8.64 was added to the reaction mixture.
  • the resulting sample was either used immediately or stored at -70 * C until electrophoresed.
  • the LYCH labelled sugars and controls were subjected to electrophoresis in a polyacrylamide gel. The separation is dependent on the molecular weight and the structure of each sugar.
  • a moving boundary (stacking) buffer system is used to give sharp bands and high resolution. This system is based on the method of Neville referred to above.
  • Electrophoresis was carried out using a resolving gel consisting of a linear gradient from 20% w/v acrylamide, 0.53% w/v, N,N' -methylenebisacrylamide (bis) to 40% w/v acrylamide, 1.06% w/v bis.
  • a stacking gel was used containing 3.0% w/v acrylamide and 0.08% w/v bis.
  • a discontinuous electrophoretic buffer system based on that described by Neville was used, but with SDS omitted. The gel was cooled by surrounding buffer at 5 to 7'C. The voltages used were 100V (constant) for 30 minutes, followed by 500V (constant) for 60 minutes, followed by 100V (constant) for 90 minutes.
  • FIG. 2 A photograph of the resulting gel illuminated from below by an ultra violet light box is shown in Figure 2.
  • a Wratten No. 8 filter was uses on the camera to reduce ultra violet and blue light coming from the light box. Bands corresponding to sugars are indicated where these are clear.
  • Preliminary experiments produced some rather fuzzy bands on the gel, which are thought possibly to be caused by impurities in the LYCH.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Diabetes (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Des substances glucidiques sont séparées ou distinguées par un procédé consistant à marquer lesdites substances à l'aide d'un réactif de marquage qui comprend un groupe hydrazide pour produire des substances marquées par fluorescence, à appliquer lesdites substances marquées à un gel électrophorétique et à faire couler le gel pour provoquer une migration différenciée de différentes substances. Le réactif de marquage préféré à l'heure actuelle est le jaune de Lucifer CH.
PCT/US1991/004555 1991-07-22 1991-07-22 Analyse de glucides et necessaire d'analyse Ceased WO1993002356A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/US1991/004555 WO1993002356A1 (fr) 1991-07-22 1991-07-22 Analyse de glucides et necessaire d'analyse
JP3513054A JPH06504363A (ja) 1991-07-22 1991-07-22 炭水化物の分析およびそのためのキット
EP91913717A EP0595806A1 (fr) 1991-07-22 1991-07-22 Analyse de glucides et necessaire d'analyse

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1991/004555 WO1993002356A1 (fr) 1991-07-22 1991-07-22 Analyse de glucides et necessaire d'analyse

Publications (1)

Publication Number Publication Date
WO1993002356A1 true WO1993002356A1 (fr) 1993-02-04

Family

ID=22225633

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/004555 Ceased WO1993002356A1 (fr) 1991-07-22 1991-07-22 Analyse de glucides et necessaire d'analyse

Country Status (3)

Country Link
EP (1) EP0595806A1 (fr)
JP (1) JPH06504363A (fr)
WO (1) WO1993002356A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6190522B1 (en) * 1998-04-24 2001-02-20 Board Of Regents, The University Of Texas System Analysis of carbohydrates derivatized with visible dye by high-resolution polyacrylamide gel electrophoresis
US6294667B1 (en) 1996-10-07 2001-09-25 Amersham International Plc Analysis of carbohydrates
WO2012010851A1 (fr) * 2010-07-23 2012-01-26 Cambridge Enterprise Limited Electrophorèse capillaire des glucides

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4163779A (en) * 1978-01-09 1979-08-07 International Diagnostic Technology, Inc. Test for quantitation of immunoglobulin and identification of abnormal immunoglobulin
US4238195A (en) * 1979-01-18 1980-12-09 Miles Laboratories, Inc. Fluorescer-labeled specific binding assays
US4473693A (en) * 1978-08-04 1984-09-25 Stewart Walter W Aminonaphthalimide dyes for intracellular labelling
US4867973A (en) * 1984-08-31 1989-09-19 Cytogen Corporation Antibody-therapeutic agent conjugates
US4876188A (en) * 1986-11-18 1989-10-24 Scripps Clinic And Research Foundation Novel immunochemical method for assaying stable glycosylated hemoglobin
US4937183A (en) * 1988-02-03 1990-06-26 Cytogen Corporation Method for the preparation of antibody-fragment conjugates
US5034514A (en) * 1986-03-17 1991-07-23 Cetus Corporation Novel cross-linking agents
US5047227A (en) * 1988-02-08 1991-09-10 Cytogen Corporation Novel and improved antibodies for site specific attachment of compounds

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4163779A (en) * 1978-01-09 1979-08-07 International Diagnostic Technology, Inc. Test for quantitation of immunoglobulin and identification of abnormal immunoglobulin
US4473693A (en) * 1978-08-04 1984-09-25 Stewart Walter W Aminonaphthalimide dyes for intracellular labelling
US4238195A (en) * 1979-01-18 1980-12-09 Miles Laboratories, Inc. Fluorescer-labeled specific binding assays
US4867973A (en) * 1984-08-31 1989-09-19 Cytogen Corporation Antibody-therapeutic agent conjugates
US5034514A (en) * 1986-03-17 1991-07-23 Cetus Corporation Novel cross-linking agents
US4876188A (en) * 1986-11-18 1989-10-24 Scripps Clinic And Research Foundation Novel immunochemical method for assaying stable glycosylated hemoglobin
US4937183A (en) * 1988-02-03 1990-06-26 Cytogen Corporation Method for the preparation of antibody-fragment conjugates
US5047227A (en) * 1988-02-08 1991-09-10 Cytogen Corporation Novel and improved antibodies for site specific attachment of compounds

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6294667B1 (en) 1996-10-07 2001-09-25 Amersham International Plc Analysis of carbohydrates
US6190522B1 (en) * 1998-04-24 2001-02-20 Board Of Regents, The University Of Texas System Analysis of carbohydrates derivatized with visible dye by high-resolution polyacrylamide gel electrophoresis
WO2012010851A1 (fr) * 2010-07-23 2012-01-26 Cambridge Enterprise Limited Electrophorèse capillaire des glucides

Also Published As

Publication number Publication date
EP0595806A1 (fr) 1994-05-11
JPH06504363A (ja) 1994-05-19

Similar Documents

Publication Publication Date Title
US5340453A (en) Analysis of carbohydrates
Liu et al. Design of 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde as a reagent for ultrasensitive determination of primary amines by capillary electrophoresis using laser fluorescence detection
JP2556659B2 (ja) 電気泳動の間に標的物質を検知する方法及び装置
Kenworthy Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy
JP3890071B2 (ja) 適合多重染料を用いる示差ゲル電気泳動
Ragland et al. Fluorometric scanning of fluorescamine-labeled proteins in polyacrylamide gels
Stephens Fluorescent thin-layer peptide mapping for protein identification and comparison in the subnanomole range
DE60016233T2 (de) Differenz-nachweismethoden unter verwendung einer vielzahl von abgestimmten farbstoffen
US5132439A (en) Protein staining compositions and methods
WO1993002356A1 (fr) Analyse de glucides et necessaire d'analyse
US5273906A (en) Protein staining compositions and methods
US5532166A (en) Quantitative retinol assay for serum and dried blood spots
US5316638A (en) Treatment of carbohydrates
Šlais et al. Fluorescein‐based pImarkers for capillary isoelectric focusing with laser‐induced fluorescence detection
Goldberg et al. Quantitation of collagen in polyacrylamide gels by fluorescent scanning of MDPF1-labeled proteins: An improvement over densitometric scanning of gels stained by coomassie blue
US5192407A (en) Means and method of detection in chemical separation procedures
Top et al. Green/red dual fluorescence detection of total protein and alkaline phosphate‐conjugated probes on blotting membranes
Lim et al. Simple and sensitive laser‐induced fluorescence detection for capillary electrophoresis and its application to protein separation
Liu et al. Home-made capillary array electrophoresis for high-throughput amino acid analysis
US20110186434A1 (en) Qualitative and/or quantitative determination of a proteinaceous molecule in a plurality of samples
Chang et al. Separation and isolation of subcellular‐size particles by electrophoresis in polymer solution using the commercial scanning apparatus
Borejdo et al. Electrophoresis and orientation of F-actin in agarose gels
Hogan et al. Indirect fluorometric detection in gel electrophoresis
Kist et al. Capillaryzone electrophoresis separation of kinins using a novel laser
US20190338131A1 (en) Hemicyanine dyes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

WWE Wipo information: entry into national phase

Ref document number: 1991913717

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1991913717

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1991913717

Country of ref document: EP