EP1649291A1 - Method for the detection of abnormally glycosylated proteins - Google Patents

Method for the detection of abnormally glycosylated proteins

Info

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Withdrawn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
German (de)
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/en
Withdrawn legal-status Critical Current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
    • G01N33/74—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

Landscapes

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

Abstract

A method of screening a sample for the presence of one or more abnormally glycosylated and/or expressed proteins comprises the steps of i) exposing said sample to two or more différent 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.

Description

METHOD FOR THE DETECTION OF ABNORMALLY GLYCOSYLATED PROTEINS
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.
Most plasma membrane and secretory proteins are complex macromolecules incorporating oligosaccharides, which play an integral role in their biological function. Consequently, abnormal glycosylation or expression of proteins is often associated with disease. For example, defects in nine genes of the N-linked glycosylation pathway are associated with serious medical conditions, including neurological dysfunction, collectively termed Congenital Disorders of Glycosylation (CDG, H. Freeze, Glycobiology 2001, 11, 129R-143R). Other conditions associated with abnormal glycosylation include Leroy disease, Wiskott-Aldrich syndrome and glycoproteinosis. Further, 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).
In addition, it is known (V. Skibeli et al., Blood 2001, 98, 3626-3634) that the glycosylated form of recombinant erythropoietin (rEPO), which is used by athletes to boost performance, differs from the natural form in that it is deficient in one type of sialic acid linkage (Neu5Ac(α2-6)Gal on N-glycans) and has supplementary poly-N- acetyl lactosamine (Gal(βl-4)GlcNAc(bl-3)) and glucosamine (GlcNAc(βl- 4)Man(βl-4)). Exogenous erythropoietin can therefore be distinguished from the endogenous protein.
Presently, methods for determining abnormal glycosylation rely on iso-electric focusing (IEF) or Western blotting of blood extracts for different glycoforms of human serum transferin (H. Stibler et al., Acta. Paediatr. Scand. Suppl., 1991, 375, 21-31; J. Jaeken et al, Clin, Chim. Acta, 1984, 144, 245 to 247; N. Seta et al., Clin. Chim. Acta 1996, 24, 131-140). Recently, however, abnormal glycosylation has been determined by lectin analysis of purified serum endocrine glycoproteins (M. Ferrari et al, Eur. J. EndocrinoL, 2001, 144, 409-416).
The detection of illicit use of rEPO is currently based on IEF of urine concentrates followed by a double blotting procedure using antibodies (F. Lasne and J. de Ceaurriz, Nature 2000, 405, 635 and F. Lasne, J. Immunol. Methods, 2001, 253 (1-2), 125-131).
However, all of the above techniques require preliminary concentration, purification or isolation of a predetermined protein and can be time consuming and/or expensive. IEF involves a multitude of steps and can often take up to three days. Further, it is not specific in that it is based on net charge of the molecule so that different defects, leading to identical net charge can have substantially similar focusing patterns.
International Patent Application WO 00/33076 discloses a method of diagnosis of human glycosylation disorders. 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.
Accordingly, in a first aspect, 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.
It will be understood that 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.
In particular, 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.
Although 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 α-2, 6- linked Neu5Ac and Maackia amurensis agglutinin (MAA), specific for α-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(αl-3/4). Suitable antibodies may be raised by any technique known to the art.
As mentioned above, the extent of binding of a particular sample to a lectin and/or antibody array, for example, 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. Thus, in a preferred embodiment 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.
It will be apparent, therefore, that 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. Thus, in one embodiment of the present invention, a body fluid sample may be directly exposed to the array.
Of course, the selection of a particular body fluid for screening will be based on the amount of glycoprotein supposed or known to be directly available therein and the sensitivity of the method utilised in the determination step. Suitable body fluids for direct analysis include urine and blood serum.
It may, however, be desirable to concentrate or purify the sample. In another embodiment, therefore, 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. In this embodiment, 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. In particular, the extent of binding of a particular sample to a particular lectin or antibody may be determined by colorimetric, fluorescence or optical techniques.
In a preferred embodiment, however, that 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.
In particular, the fingerprint or signature may be interrogated by an evanescent optical technique. Preferably, therefore, 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.
Preferably, the optical interrogation utilises a flow technique. Suitable apparatus, therefore, 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.
In particular, the comparison may be made by reference to a library database including fingerprint data from a number of control samples. Preferably, the- - comparison is computer aided. In this embodiment, the fingerprint data deposited in the database need only record salient features characterising health and/or abnormal glycosylation.
An exposure of the 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. In particular, 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.
It will appreciated from the foregoing, that 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. Thus 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.
In addition, 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.
In a second aspect, therefore, 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.
It will be understood that the 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.
Embodiments of the second aspect of the present invention will be apparent from the above and the claims appended hereto.
As mentioned above, 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. In a third aspect, therefore, 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.
Embodiments of the third aspect of the present invention will also be apparent from the above and the claims appended hereto.
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.
In a further aspect, 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. Additionally, 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. Preferably, the solid support surface comprises an SPR, MCLW or DCLW chip. In a preferred embodiment, suitable for detecting the illicit use of a performance enhancing drug, such as erythropoietin, the kit may comprise only lectins.
The supplied information may also include information relating binding patterns and/or characteristics to candidate disease states. In particular, the additional information may categorise certain binding patterns and/or characteristics according to likely medical condition.
The present invention will now be described by reference to the following examples and drawings in which 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 graph showing the dissociation characteristics of human EPO/NESP mixtures from MAA according to Figure 5 relative to the proportion of EPO; Figures 7 a) and b) are graphs showing the ratio of response to the SNA/MAA array according to Figure 5 of human EPO/NESP mixtures and human EPO/human fetuin mixtures relative to the proportion of EPO; Figure 8 is a SPR sensor-gram illustrating the interaction of a urine sample respectively with the SNA/MAA array; and Figures 9 a) and b) are graphs highlighting the screening of human blood serum samples for alcoholism or related diseases using a four-fold lectin array.
Referring now to Figure 1, the putative response of four lectins (1 to 4) to a control sample a) and two samples b) and c) exhibiting an abnormal glycosylation disorder confer a fingerprint or signature to each sample (al, a2, a3, a4 etc). The fingerprint or signature of sample b), when compared to that of the control sample, indicates the disorder although it is not apparent from its interaction with lectin 2 alone.
Having regard to Figures 2 to 8, experiments were performed on a BIAcore® 2000 four flow channel SPR instrument equipped with BIAevaluation software 3.0. Lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin (Sigma, Madrid) were each immobilised to part of an SPR chip (carboxymethylated dextran surface, standard density CM5 sensorchip, Pharmacia Biosensor AB, Uppsala, Sweden) for registration with each channel using known amine coupling techniques (S.R. Haseley et al., Anal. Biochem., 1999, 271, 203-210). Table 1 summarises the immobilisation parameters for the chip.
Table 1 Example 1 rEPO expressed in CHO cells (European Pharmacopeia, France) was made up to 5 μgml"1 in distilled water. Bovine fetuin (Sigma, Madrid) was made up to lOμgmi"1 in distilled water. The interactions of 35 μl 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 CaCl2 and 2 mM MgCl2 in distilled water).
Referring now to Figure 2 a), the binding of rEPO to MAA is much greater than to SNA. rEPO, in common with the natural form of EPO, has a significant number of α- 2,3-Neu5Ac linkages - specifically recognised by MAA but, in contrast to the natural form of EPO, only a single α-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 α-2,6-Neu5Ac linkage.
Referring now to Figure 2 b), the binding of bovine fetuin, which is similar to human EPO in that it comprises significant numbers of both α-2,6-Neu5Ac and α-2,3- Neu5Ac linkages, to MAA is gradual and low. By contrast, the binding to SNA is rapid and sustained at high levels.
It will be appreciated that concentration effects and the presence of linkages in common do not permit one or other of these lectins alone to definitively indicate whether the sample contains exogenous protein in addition to the endogenous protein. However, these results suggest that the interaction of a sample with two or more lectins will, however, allow exogenous protein to be distinguished - even in the presence of the natural form.
Further, the presence of 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.
Example 2
Referring now to Figures 3 a) to c), the binding (after 7 min. of 5 μl min"1 running buffer TRIS, pH 7.2, 150 mM NaCl, 2 mM CaCl2 and 2mM MgCl2) of each of human EPO (1 μl from stock in 200 μl, b) and human fetuin (7.5 μgml"1 solution, c), to a similar chip is greater to SNA than to MAA. However, the binding of NESP (8 μl from stock in 200 μl, a), a recombinant form of EPO, to SNA is almost negligible when compared to MAA.
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.
Referring now to Figure 5, the interaction of each lectin with mixtures a) to c) of varying ratios of human EPO to NESP, are of interest not only in the ratio of the level of binding but also in the dissociation phase of each response. It will be noted that the fingerprints and the dissociation curves are different for each mixture as well as for the pure samples.
The linear relationship between the amount of EPO and ka for MAA and/or SNA as well as SNA/MAA binding ratio also suggests that the method may, in combination with other techniques, enable quantitative determination of rEPO in a sample.
Table 2 compares the ratio of response of the lectins and the rate of dissociation kd 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. These results suggest that NESP and, indeed, human fetuin can be determined in the presence of human EPO.
Table 2 Sensitivity experiments performed on the chip showed that the lower detection limit of rEPO by MAA was in the range of 5 ngml-1 (160 pmol.l"1) which is comparable to the reference solution used for IEF (380 pmol.l"1).
Referring now to Figure 8, the results of the interaction of a control urine sample, concentrated 20-fold, is shown. Here, the chip has in addition to a blank (-x- line) surface, has a surface in which the dextran surface is not activated for lectin immobilisation (-+-). As may be seen, glycoprotein interactions with MAA and SNA are present. Reference to values (0 to 5.3 pmol.l"1) published for EPO in urine suggest the need for a 200 fold increase in sensitivity. It is envisaged, however, that routine optimisation of the technique (by selection of lectins and running buffers) and/or detection method (for example, light or emitted from the array through fluorescent tagging) will enable urine samples to be directly screened without the need for a concentration or isolation step.
For the determination of abuse of rEPO large arrays including MAA, SNA and lectins specific for poly N-acetyl lactosamine repeat units (Gal(βl-4)GlcNAc(βl-3) or lectins specific for bisecting GlcNAc (GlcNAc(βl-4)Man(βl-4) are expected to offer significant improvements. Alternatively or additionally, arrays including lectins or antibodies specific for tetraantennary glycan moieties found mostly with the recombinant form may also be of value.
Table 3 Having regard now to Figures 9 a) and b), similar experiments were performed on 24 human blood serum samples using an SPR chip including an array of four lectins Bauhinia purpureus agglutinin (BPA), Maackia amurensis agglutinin, Sambucus nigra agglutinin and Ricinus communis 120 agglutinin (RCA12o). Table 3 summarises the immobilisation responses of each lectin on the chip.
Example 3
Half of the serum samples were obtained from patients with alcoholism or related diseases, and so contained high (H1-H12) levels of carbohydrate deficient transferrin (CDT) relative to the total content of transferrin. The other half of the serum (control) samples had normal (low, LI to LI 2) levels of carbohydrate deficient transferrin (CDT) relative to the total content of transferrin.
The serum samples were simply diluted (1:100) in running buffer (10 mM Tris.HCl, pH 7.0; 150 mM NaCl; 2 mM CaCl2 and 2 mM MgCl2) and the interaction of each diluted sample with the chip was studied at various time (t) intervals
At t = 150 s 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.
For example, 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 (Figure 9 a) 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 (Figure 9 b) 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.
In particular, it is envisaged that 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.
It is also envisaged that a wide variety of other lectins may be used and that the interaction of a sample with large numbers of lectins will not be unduly complicated - especially where analysis is computer-aided.

Claims

1. 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.
2. A method according to Claim 1, in which the sample comprises a human or animal body fluid.
3. A method according to Claim 2, in which the body fluid sample is directly exposed to said two or more lectins and/or antibodies.
4. A method according to any preceding Claim, in which the determination is performed in real time.
5. A method according to Claim 4, in which the sample is simultaneously exposed to an array of said two or more lectins and/or antibodies immobilised on a solid support surface.
6. A method according to any of Claims 3 to 5, in which the determination is performed by an evanescent optical technique.
7. A method according to Claim 6, in which said apparatus detects light reflected from at the solid support surface.
8. A method according to any preceding Claim, in which said two or more lectins and/or antibodies comprise only lectins.
9. A method according to any preceding Claim, in which the lectins are specific for sialic acid, galactose, mannose, glucosamine or fucose containing oligosaccharides.
10. A method according to Claim 9, in which the lectins comprise Sambucus nigra agglutinin and/or Maackia amurensis agglutinin.
11. A method according to any preceding Claim, in which the protein comprises recombinant erythropoietin, chorionic gonadotropin or human growth hormone.
12. A method according to any of Claims 1 to 10, in which the protein comprises a transferrin.
13. A method for determining use of a glycoprotein drug in a mammal 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 of said lectins and/or antibodies and iii) comparing the extent of binding of said at least two lectins and/or antibodies to that of a control sample.
14. A method according to Claim 13, comprising the features of any one of Claims 2 to 11.
16. 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 binding pattern 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 to that of a control sample.
17. A method according to Claim 16, comprising the features of any one of Claims 2 to 10 or Claim 12.
18. A kit of parts for use in the method of any preceding Claim, 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.
19. A kit according to Claim 18, further comprising an SPR, MCLW or DCLW chip.
20. A kit according to Claim 18 or Claim 19, in which the information includes information relating binding patterns and/or characteristics to candidate disease states.
21. A kit according to any of Claims 18 to 20, comprising only lectins.
22. A kit according to any of Claims 18 to 21, in which the lectins comprise lectins specific for sialic acid, galactose, mannose, glucosamine or fucose moities in the protein.
23. A kit according to Claim 22, in which the lectins comprise Sambucus nigra agglutinin and/or Maackia amurensis agglutinin.
EP04743670A 2003-08-05 2004-08-05 Method for the detection of abnormally glycosylated proteins Withdrawn EP1649291A1 (en)

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

Publications (1)

Publication Number Publication Date
EP1649291A1 true EP1649291A1 (en) 2006-04-26

Family

ID=27839646

Family Applications (1)

Application Number Title Priority Date Filing Date
EP04743670A Withdrawn EP1649291A1 (en) 2003-08-05 2004-08-05 Method for the detection of abnormally glycosylated proteins

Country Status (5)

Country Link
EP (1) EP1649291A1 (en)
AU (1) AU2004264053A1 (en)
CA (1) CA2534815A1 (en)
GB (1) GB2404734A (en)
WO (1) WO2005015240A1 (en)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
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 (en) * 2008-01-14 2013-03-27 开物科技股份有限公司 Electronic testing device and testing method
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 (en) * 2014-08-05 2015-06-03 首都医科大学附属北京佑安医院 Chemical luminescent protein chip, kit and detection method for detecting fucose index of seroglycoid
FI20155280A7 (en) 2015-04-15 2016-10-16 Medicortex Finland Oy Prognostic and diagnostic glycan-based biomarkers of brain damage
US20210311043A1 (en) * 2020-04-06 2021-10-07 Medicortex Finland Oy Method for determining a lectin-binding glycan indicative to traumatic brain injury

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (en) * 1984-08-07 1994-12-21 協和メデックス株式会社 Cancer measurement kit
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
JP3950481B2 (en) * 1996-09-06 2007-08-01 エール ユニバーシティ Prenatal screening for Down syndrome using hyperglycosylated gonadotropins
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
EP1495323A2 (en) * 2002-04-05 2005-01-12 University Of Georgia Research Foundation, Inc. Method for cleaving and deglycosylating antibodies to promote ligand binding

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2005015240A1 *

Also Published As

Publication number Publication date
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

Similar Documents

Publication Publication Date Title
EP1583968B1 (en) A serum marker for measuring liver fibrosis
EP0189388B1 (en) Diagnostic method for diseases having an arthritic component
EP2357475B1 (en) Method for measuring cystatin c in human body fluid
EP3775896B1 (en) Lateral flow immunoassay strip device
Skarulis et al. Glycosylation changes in human chorionic gonadotropin and free alpha subunit as gestation progresses
JP2007163505A (en) Prenatal screening for Down syndrome using hyperglycosylated gonadotropins
EP2006685A1 (en) Immune agglutination reaction reagent kit and method of assaying antigen
CN110028539A (en) The bionical sugar of isotope labelling or sugared group, preparation method and application
JP4711190B2 (en) Test method for glycosylation disorder
EP1649291A1 (en) Method for the detection of abnormally glycosylated proteins
KR20140011760A (en) Antibody-lectin sandwich assay for the measurement of breast cancer marker
JP2007514438A (en) Method for analyzing glycated molecules
DE19637418A1 (en) Homogeneous detection methods for the determination of subpopulations of an analyte
US20240077486A1 (en) Flow Cytometry Platform for the Detection of Glycosylated Proteins in a Clinical Sample
KR101143891B1 (en) A marker for the diagnosis of cancers by using aberrant glycosylation of protein
CN109580931A (en) A kind of α 1- microglobulin detection kit
US8697384B2 (en) YKL-40 as a general marker for non-specific disease
KR20050118178A (en) Latex reagent for adiponectin and method of adiponectin analysis
CN110133270A (en) A kind of enhancing immunosupress is than turbid antibody screening reagent and its making and use method
DE69821485T2 (en) TEST FOR CARBOHYDRATE-FREE TRANSFERRIN
KR101100809B1 (en) Peptide Markers for Cancer Diagnosis and Cancer Diagnosis Methods Using the Same
Das et al. Should we use carbohydrate deficient transferrin as a marker for alcohol abusers?
Lönnberg Membrane-Assisted Isoform ImmunoAssay: Separation and determination of protein isoforms
Gornik et al. Standardizing Lectin-ELISA for Routine Screening of Serum Transferrin Glycosylation
Dong The developments and applications of LC-MS based quantitative methods for glycomics

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20060206

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

17Q First examination report despatched

Effective date: 20070423

18W Application withdrawn

Effective date: 20070427