EP1649291A1 - Verfahren zum nachweis von abnormal glykosylierten proteinen - Google Patents

Verfahren zum nachweis von abnormal glykosylierten proteinen

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Publication number
EP1649291A1
EP1649291A1 EP04743670A EP04743670A EP1649291A1 EP 1649291 A1 EP1649291 A1 EP 1649291A1 EP 04743670 A EP04743670 A EP 04743670A EP 04743670 A EP04743670 A EP 04743670A EP 1649291 A1 EP1649291 A1 EP 1649291A1
Authority
EP
European Patent Office
Prior art keywords
lectins
sample
antibodies
binding
extent
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Withdrawn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
EP04743670A
Other languages
English (en)
French (fr)
Inventor
Simon Richard Haseley
Martin Bernard Mcdonnell
Debbie Brenda Padgen
J. Pharm. Res. Unit Inst. of Med. Res. Segura
R. Pharm. Res. Unit Inst. of Gutierrez-Gallego
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.)
UK Secretary of State for Defence
Original Assignee
UK Secretary of State for Defence
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 UK Secretary of State for Defence filed Critical UK Secretary of State for Defence
Publication of EP1649291A1 publication Critical patent/EP1649291A1/de
Withdrawn legal-status Critical Current

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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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/746—Erythropoetin
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • 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
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
    • G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin
    • 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
    • G01N2333/575—Hormones
    • G01N2333/61—Growth hormones [GH] (Somatotropin)

Definitions

  • the present invention relates to a method of screening a sample for the presence of one or more abnormally glycosylated and/or expressed proteins.
  • the present invention is particularly, although not exclusively, concerned with screening a biological sample, for example a body fluid sample, for the presence of abnormally O- and/or N-glycosylated proteins, which may be indicative of disease or of substance abuse in an individual.
  • CDG Congenital Disorders of Glycosylation
  • Other conditions associated with abnormal glycosylation include Leroy disease, Wiskott-Aldrich syndrome and glycoproteinosis.
  • diseases resulting from exposure to some external factor such as alcoholism, rheumatoid arthritis and cancer are also associated with abnormal glycosylation of proteins (see for example, G. Durand and N. Seta, Clin. Chem. 2000, 46, 795-805).
  • IEF infrared emission spectroscopy
  • a first, reagent for example a lectin, binds with an oligosaccharide moiety present in a sample from an individual having a glycosylation disorder and not present in a sample from a healthy individual.
  • a second, reagent binds with an oligosaccharide present in a healthy individual but not present in an individual having a glycosylation disorder.
  • a glycosylation disorder is indicated through binding of the first reagent and/or non-binding of the second reagent.
  • the method is, however, dependent on the presence or absence of a target oligosaccharide moiety in the sample compared with a control sample. It is, therefore, unable to detect glycosylation disorders characterised by the relative extent and/or ratio of oligosaccharide moieties (or epitopes). There is consequently a need for an improved method for determining abnormal glycosylation or substance abuse in individuals.
  • the present invention generally seeks to provide an improved method for screening a sample, in particular a body fluid sample, for one or more abnormally glycosylated proteins.
  • the present invention also aims to improve diagnosis of disease exhibiting abnormal glycosylation and to improve testing for performance enhancing protein drugs in mammals.
  • the present invention also seeks to provide an improved method for determining the presence of an abnormally expressed protein in a body fluid sample.
  • Such a method maybe of value to the diagnosis of disease, such as Alzeihemer's, BSE and the like.
  • the present invention provides a method of screening a sample for the presence of one or more abnormally glycosylated and/or expressed proteins comprising the steps of i) exposing said sample to two or more different lectins and/or antibodies ii) determining the extent of binding of said sample to at least two of said lectins and/or antibodies and iii) comparing the determined extent of binding to said at least two lectins and/or antibodies with that of a control sample.
  • the method is preferably used for screening biological samples, i.e. those taken from the human or animal body. However, it may also be used to screen other samples such as those used during laboratory experimentation.
  • a particular sample may be exposed to the lectins and/or antibodies separately and sequentially. In a preferred embodiment, however, the sample is exposed to the said two or more lectins and/or antibodies simultaneously.
  • the lectins and/or antibodies may be immobilised on a solid support surface and arranged in an array and the sample sequentially and/or simultaneously exposed to them.
  • the determination of the extent of binding to a selection of different lectins and/or antibodies is of critical importance to the present invention, there is no requirement that more than two, or any one particular lectin or antibody is used. All that is required is that a pattern of the extent of binding is determined for a particular sample. Preferably, however, the extent of binding is determined for more than two lectins and/or antibodies.
  • Suitable lectins include those that are specific for sialic acid (Neu5Ac), galactose, mannose, glucosamine and fucose containing oligosaccharides. Amongst these, the commercially available lectins Sambucus nigra agglutinin (SNA), specific for ⁇ -2, 6- linked Neu5Ac and Maackia amurensis agglutinin (MAA), specific for ⁇ -2, 3-linked Neu5Ac are preferred. Other suitable, lectins include Tetragonolobus purpureaus (TLP), Anguila anguila (AA) and Ulex europaeus I agglutinin (UEA) which are specific for Fuc( ⁇ l-3/4). Suitable antibodies may be raised by any technique known to the art.
  • the extent of binding of a particular sample to a lectin and/or antibody array follows a unique pattern.
  • the pattern which can be thought of as a "fingerprint” or “signature” of the sample on the array, characterises the glycosylation environment of the sample, each lectin, for example, being specific to a group of related oligosaccharide epitopes.
  • the fingerprint or signature of a particular sample may be expressed by one or more ratios of extent of binding to different lectins and/or antibodies.
  • the comparison with the control sample compares one or more ratios (A/B, A/C, B/C etc.) of extent of binding to different lectins and/or antibodies (A, B, C) for each sample.
  • the method is particularly suited to screening complex samples - which may be expected to exhibit binding to most or all of the lectins and/or antibodies.
  • the method can avoid the need for concentration, purification and isolation of a previously identified protein since, normally, it is the comparison of the fingerprint or signature with that of a control sample that is indicative of the presence of an abnormally glycosylated or expressed protein.
  • a body fluid sample may be directly exposed to the array.
  • Suitable body fluids for direct analysis include urine and blood serum.
  • the method of the present invention comprises the preliminary step of isolating one or more proteins from the sample by capture with an antibody or antibodies.
  • the isolation of protein or proteins minimises unwanted complications in the determination step - especially where the concentration of the proteins approaches the limits of detection and quantification.
  • the fingerprint or signature of a particular sample may be interrogated by any suitable means determining the extent of binding of the sample to said at least two lectins and/or antibodies.
  • the extent of binding of a particular sample to a particular lectin or antibody may be determined by colorimetric, fluorescence or optical techniques.
  • the fingerprint or signature is interrogated by a technique allowing real-time monitoring of the binding of the sample to the array.
  • Such techniques also allow kinetic characteristics such as binding or dissociation rates, to be determined providing further information as to the presence of abnormal glycosylated or expressed proteins.
  • the fingerprint or signature may be interrogated by an evanescent optical technique.
  • the solid support and array together comprise an optical biosensor.
  • Suitable solid supports include surface plasmon resonance (SPR) chips and/or metal or dye clad leaky waveguide (MCLW/DCLW) chips, such as those described in our international applications WO 99/44042 and PCT/GB02/045045.
  • the optical interrogation utilises a flow technique.
  • Suitable apparatus include ordinary and optimised surface plasmon resonance apparatus described in International Patent Application WO 01/42768.
  • the comparison of the interrogated fingerprint or signature with that of the control sample does not necessarily require that the investigator himself make an exposure of the control sample to an identical array.
  • the comparison may be made by reference to a library database including fingerprint data from a number of control samples.
  • the- - comparison is computer aided.
  • the fingerprint data deposited in the database need only record salient features characterising health and/or abnormal glycosylation.
  • control sample may, however, be preferred in lectin-only arrays where the relatively weak lectin-glycoprotein interaction allows easy regeneration.
  • the control sample may be derived from a healthy individual or an individual exhibiting abnormal glycosylation.
  • the control sample may be one or more earlier samples taken from the same individual. Such a control sample is especially useful for monitoring continuation of substance abuse or the progress of disease and/or treatment.
  • the present invention provides an improved method of screening samples, in particular body fluid samples, in that it compares the ratio of binding to two or more lectins and/or antibodies.
  • the method is not dependent on the absence of certain epitopes in one or other of the samples and is less prone to false positives.
  • real-time monitoring based on optical techniques allows rapid screening of complex samples in a single exposure of the sample to a lectin and/or antibody array.
  • Such techniques allow simultaneous determination of binding to each lectin and/or antibody in an array and may offer additional information which may characterise the absence or presence of an abnormally glycosylated or expressed protein in a sample.
  • the method of the present invention may also be used to screen a body fluid sample for indication of use or abuse of glycoprotein drugs in humans or animals.
  • the present invention provides a method for determining use of a glycoprotein drug in a human or animal comprising the steps of i) taking a body fluid sample ii) exposing said sample to two or more different lectins and/or antibodies iii) determining the extent of binding of said sample to at least two lectins and/or antibodies and iii) comparing the determined extent of binding to said at least two lectins and/or antibodies with that of a control sample.
  • glycoprotein drug is synthetic in origin and differs from the endogenous form in, for example, its glycosylation environment.
  • the method is particularly suitable for determining abuse or illicit use of recombinant performance enhancing protein drugs, such as recombinant forms of erythropoietin, chorionic gonadotropin or human growth hormone.
  • the method provides for detection of exogenous protein through pattern recognition indicating the presence of abnormal glycoforms of the protein - even in the presence of endogenous protein.
  • Exposure of a urine sample to a lectin-only array, for example, and comparison of the interrogated fingerprint in relation and/or other indicia with that of a control sample as explained above can provide conclusive evidence of use.
  • the fingerprint or signature of a particular sample may be informative as to a particular disease state in an individual when compared to that of a sample from a healthy individual.
  • the present invention provides a method for the diagnosis of acquired or inherited glycosylation disorders comprising the steps of i) taking a body fluid sample ii) exposing said sample to two or more different lectins and/or antibodies iii) determining the extent of binding of said sample to at least two lectins and/or antibodies and iii) comparing the determined extent of binding to said at least two lectins and/or antibodies with that of a control sample.
  • the method in this aspect of the present invention may, for example, be particularly suitable for diagnosis of disease in neonates, children or adults.
  • the method is not necessarily limited to medical conditions which are attributable to abnormal glyscosylation but may also be of value to conditions accompanied by abnormal glycosylation.
  • the present invention provides a kit of parts for use in the aforementioned methods comprising one or more lectins and/or antibodies and a control sample and/or information relating to normal or expected glycosylation binding patterns and/or characteristics for an individual type.
  • the lectins and/or antibodies may be supplied in vials or a suitable container or containers.
  • a solid support surface may be supplied for attachment of a selection of lectins and/or antibodies by any of the usual methods known to the art.
  • the solid support surface comprises an SPR, MCLW or DCLW chip.
  • the kit may comprise only lectins.
  • the supplied information may also include information relating binding patterns and/or characteristics to candidate disease states.
  • the additional information may categorise certain binding patterns and/or characteristics according to likely medical condition.
  • Figure 1 is a putative bar graph illustrating the fingerprint or signature of samples (a, b, c) on a four-fold lectin array
  • Figures 2 a) and b) are SPR sensor-grams illustrating respectively the binding of buffered rEPO and bovine fetuin solutions to a SNA/MAA array
  • Figures 3 a), b) and c) are SPR sensor-grams illustrating respectively the binding of a buffered solution of NESP (a rEPO), human EPO and human fetuin to a SNA/MAA array
  • Figure 4 is a bar graph showing a fingerprint or signature for the samples of Figures 3 a) " to c)
  • Figures 5 a) to d) are SPR sensor-grams illustrating the binding of buffered solutions of mixtures of human EPO/NESP and human EPO/human fetuin on a SNA/MAA array
  • Figure 6 is a putative bar graph illustrating the
  • Example 1 rEPO expressed in CHO cells (European Pharmacopeia, France) was made up to 5 ⁇ gml "1 in distilled water.
  • Bovine fetuin (Sigma, Madrid) was made up to lO ⁇ gmi "1 in distilled water.
  • the interactions of 35 ⁇ l of each of the rEPO and bovine fetuin solution with the chip was studied in running buffer (pH 7.5; 100 mM HEPES, 150 mM NaCl, 2 mM CaCl 2 and 2 mM MgCl 2 in distilled water).
  • rEPO in common with the natural form of EPO, has a significant number of ⁇ - 2,3-Neu5Ac linkages - specifically recognised by MAA but, in contrast to the natural form of EPO, only a single ⁇ -2,6-Neu5Ac linkage - specifically recognised by SNA.
  • the binding of rEPO with MAA shows a rapid uptake of the glycoprotein to the lectin, which gradually levels off. A low level of disassociation of the glycoprotein follows - the residual level of binding remaining high.
  • the binding of rEPO with SNA include an initial uptake of the glycoprotein, which almost immediately levels off. Residual binding of the glycoprotein tends to zero even though it contains one ⁇ -2,6-Neu5Ac linkage.
  • bovine fetuin which is similar to human EPO in that it comprises significant numbers of both ⁇ -2,6-Neu5Ac and ⁇ -2,3- Neu5Ac linkages, to MAA is gradual and low.
  • the binding to SNA is rapid and sustained at high levels.
  • rEPO in a sample will be apparent not just through analysis of the ratio of MAA/SNA binding compared with that from a healthy mammal but also through comparison of the analysis of uptake, equilibrium and/or dissociation characteristics of each sample.
  • Figure 4 shows the fingerprint or signature of each sample in relation to both lectins. As may be seen the ratio of binding having regard to both lectins distinguishes the samples from each other.
  • Table 2 compares the ratio of response of the lectins and the rate of dissociation k d for each sample. As is clear, especially in regard to Figures 6 and 7 a), the ratio of binding response SNA/MAA and the rate of dissociation from MAA varies linearly with the amount of EPO in the mixture.
  • arrays including MAA, SNA and lectins specific for poly N-acetyl lactosamine repeat units (Gal( ⁇ l-4)GlcNAc( ⁇ l-3) or lectins specific for bisecting GlcNAc (GlcNAc( ⁇ l-4)Man( ⁇ l-4) are expected to offer significant improvements.
  • arrays including lectins or antibodies specific for tetraantennary glycan moieties found mostly with the recombinant form may also be of value.
  • the serum samples were simply diluted (1:100) in running buffer (10 mM Tris.HCl, pH 7.0; 150 mM NaCl; 2 mM CaCl 2 and 2 mM MgCl 2 ) and the interaction of each diluted sample with the chip was studied at various time (t) intervals
  • the high (between 3.5 to 9.2 % transferrin) and low (between 2.0 to 2.6 % transferrin) CDT containing samples can be reliably distinguished by examining one or more of the order of response in the lectin array as well as the magnitude and relative magnitude of these responses.
  • high CDT samples were found to have, in general, an order of response Ch: 3, 4, 1, 2 or 3, 2, 4, 1 whereas the majority of low CDT samples gave an order of response 3, 4, 2, 1.
  • the difference (D, 4-2) in response of SNA and MAA to high CDT samples was generally high or negative compared with low CDT samples.
  • the ratio (R, 3/4) in response of RCA120 to SNA to low CDT samples tended to be lower than high CDT samples.
  • a plot of difference D, 4-2 against R, 3/4 shows tight clustering of the majority of low CDT samples and two distinct clusters of high CDT samples.
  • a plot of ratio R, 3/4 against ratio R, 4/2 similarly reveals tight clustering of low CDT samples.
  • the present invention may be suitable for rapid recognition of a wide variety of inherited or congenital disorders associated with abnormal glycosylation. Blood serum or urine may be screened for other medical conditions, not mentioned above.
  • the method is suitable for prenatal screening for Down's syndrome in that concentrations of human chorionic gonadotropin (hCG) or sub-units thereof are high in the serum and urine of pregnant women.
  • hCG human chorionic gonadotropin

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EP04743670A 2003-08-05 2004-08-05 Verfahren zum nachweis von abnormal glykosylierten proteinen Withdrawn EP1649291A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0318296A GB2404734A (en) 2003-08-05 2003-08-05 A method of screening a sample for abnormally glycosylated and/or expressed proteins
PCT/GB2004/003385 WO2005015240A1 (en) 2003-08-05 2004-08-05 Method for the detection of abnormally glycosylated proteins

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EP1649291A1 true EP1649291A1 (de) 2006-04-26

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EP04743670A Withdrawn EP1649291A1 (de) 2003-08-05 2004-08-05 Verfahren zum nachweis von abnormal glykosylierten proteinen

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EP (1) EP1649291A1 (de)
AU (1) AU2004264053A1 (de)
CA (1) CA2534815A1 (de)
GB (1) GB2404734A (de)
WO (1) WO2005015240A1 (de)

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Publication number Priority date Publication date Assignee Title
WO2002071067A1 (en) * 2001-03-07 2002-09-12 Bio-Rad Laboratories, Inc. Assay system for simultaneous detection and measurement of multiple modified cellular proteins
CN101487844B (zh) * 2008-01-14 2013-03-27 开物科技股份有限公司 电子检验装置及检测方法
CA2764153C (en) 2009-06-02 2021-07-27 The Board Of Regents Of The University Of Texas System Identification of small molecules recognized by antibodies in subjects with neurodegenerative diseases
US8759259B2 (en) 2009-10-16 2014-06-24 The Board Of Regents Of The University Of Texas System Compositions and methods for producing cyclic peptoid libraries
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
FI20155280A7 (fi) 2015-04-15 2016-10-16 Medicortex Finland Oy Aivovamman prognostisia ja diagnostisia glykaanipohjaisia biomarkkereita
US20210311043A1 (en) * 2020-04-06 2021-10-07 Medicortex Finland Oy Method for determining a lectin-binding glycan indicative to traumatic brain injury

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US4939240A (en) * 1983-03-04 1990-07-03 Health Research, Inc. Monoclonal antibodies to human breast carcinoma cells and their use in diagnosis and therapy
JPH06105257B2 (ja) * 1984-08-07 1994-12-21 協和メデックス株式会社 癌の測定用キット
GB8726271D0 (en) * 1987-11-10 1987-12-16 Univ London Protein glycosylation assay
US4996298A (en) * 1988-04-20 1991-02-26 New England Deaconess Hospital Corporation Marker for colorectal carcinoma and methods of detecting the same
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AUPO943297A0 (en) * 1997-09-24 1997-10-16 University Of Melbourne, The Diagnostic test for alzheimer's disease
WO2002071067A1 (en) * 2001-03-07 2002-09-12 Bio-Rad Laboratories, Inc. Assay system for simultaneous detection and measurement of multiple modified cellular proteins
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GB0318296D0 (en) 2003-09-10
AU2004264053A1 (en) 2005-02-17
GB2404734A (en) 2005-02-09
WO2005015240A1 (en) 2005-02-17
CA2534815A1 (en) 2005-02-17

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