WO2003058249A1 - Procede de quantification de niveaux de proteines dans des echantillons biologiques - Google Patents

Procede de quantification de niveaux de proteines dans des echantillons biologiques Download PDF

Info

Publication number
WO2003058249A1
WO2003058249A1 PCT/GB2003/000112 GB0300112W WO03058249A1 WO 2003058249 A1 WO2003058249 A1 WO 2003058249A1 GB 0300112 W GB0300112 W GB 0300112W WO 03058249 A1 WO03058249 A1 WO 03058249A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
protein
unlabelled
disease
samples
Prior art date
Application number
PCT/GB2003/000112
Other languages
English (en)
Inventor
Mikhail Soloviev
Original Assignee
Oxford Glycosciences (Uk) Ltd
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 Oxford Glycosciences (Uk) Ltd filed Critical Oxford Glycosciences (Uk) Ltd
Priority to AU2003201657A priority Critical patent/AU2003201657A1/en
Publication of WO2003058249A1 publication Critical patent/WO2003058249A1/fr

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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins

Definitions

  • the invention disclosed provides a novel method for the quantitation of differential levels of proteins using unlabelled samples in particular using protein binding arrays.
  • the method is useful for assessing differential expression or post translational modification of proteins, and in diagnosis and screening where differential expression or post translational modification of proteins is an indicator of the diseased state.
  • the method can be extended to permit the use of generic arrays.
  • a cell can express a large number of different proteins, and the expression patterns (the number of proteins expressed and the expression levels) vary in different cell types, explaining why different cells perform different functions. Since many diseases associate with, or even result from a change in the protein expression pattern, comparing protein expression patterns between normal and disease conditions can reveal proteins whose changes are critical in the disease and thus can identify proteins which are of therapeutic value or which aid diagnosis. Methods of detecting protein expression profiles also have important applications in, for example and without limitation, tissue typing, forensic identification, toxicology testing and clinical diagnosis. Detecting patterns of post translational modification can convey similarly useful information.
  • Microarray formats for the quantitative detection of proteins have been developed for diagnosis (Ekins & Chu, 1999, Trends in Biotechnol. 17:217-8) and protein-protein interaction discovery (Walter et al., 2000, Curr. Opin. Microbiol. 3:298-302; and US 6,197,599).
  • development of a microarray affinity capture system for semi-quantitative or quantitative proteomics similar to those used for semi-quantitative mRNA expression analyses (Schena et al., 1998, Trends in Biotechnol. 16:301-6) has lagged behind.
  • mRNA microarrays are well advanced and identification of the differential sequences is straightforward, there is a large inconsistency between mRNA and protein levels (Gygi, SP et al., 1999, Mol. Cell Biol. 19:1720-30) and since proteins are the primary agents responsible for disease and drug responses, predicting protein presence and amount by measurement of mRNA can lead to significant errors, sometimes as much as 20-fold or larger. Even more important is that activities of many proteins can be grossly affected by post-translational modifications such as phosphorylation. A nucleic acid array cannot detect such effects.
  • proteomics techniques such as 2D-gel electrophoresis require complex procedures to identify differentially expressed proteins (Parekh et al.2000, Journal of Commercial Biotechnology 6:284-291). Most significantly, low abundance proteins can escape detection due to limitations in visualizing the proteins present in the 2D-gel.
  • a microarray format, high throughput system for quantitative direct detection of proteins such as is required in proteomics applications is thus highly desirable for, without limitation, diagnosis, pharmacoproteomics, identification of markers of disease and drug target discovery.
  • Protein binding arrays known in the art suffer from multiple drawbacks (see Kodadek, T., 2001 , Chemistry & Biology 8:105-115 for a discussion). In particular, there are nonspecific binding and background issues where high levels of label e.g. fluorescent label, can obscure specific binding. There are also sample labelling issues where the inherent heterogeneity of proteins, polypeptides and peptides means that they can all take up label to different extents making comparison of expression levels impossible (Abbot, A., 2002, Nature 415:112-114). Inherent in labelling a sample is the need to purify the sample free from unbound reactive label.
  • protein labelling results in chemical modification of the surface of the proteins and may decrease the affinity of an antibody-protein interaction or a protein-protein or protein-nucleic acid interaction.
  • biomolecules of interest are small polypeptides or short peptides the introduction of a chemical modification in the form of a label may interfere such that total disruption of the binding of said biomolecules to cognate capture agents or other binding partners occurs.
  • This is unlike the labelling of nucleic acids, where a label is usually introduced via an enzymatic reaction into a defined position (such as the 3'- or 5'-end of a nucleic acid) and hence do not disrupt the interaction between complementary chains.
  • nucleic acid arrays Another major difference between nucleic acid arrays and protein arrays is that for any arrayed nucleic acid, the affinity of its interaction with a corresponding complementary strand is a product of length up to a plateau value. The affinity of interaction is thus similar for all nucleic acid fragments of similar lengths.
  • antibodies are inherently heterogeneous and hence their affinities for their respective antigens are often very different irrespective of size. In the context of an antibody array, these differences in affinities can result in a wide range of signal intensities even if equimolar amounts of a labelled sample are added to an array of cognate antibodies.
  • highly abundant proteins may fully saturate the immobilized antibody and mask any differential binding signal between two samples.
  • RIA radioimmunoassay
  • RIA utilises the competitive binding between pure, labelled antigen and pure unlabelled antigen for an antibody which specifically recognises said antigens to determine the amount of antigen in a sample
  • the latter method requires the use of a purified antigen labelled with minimal alteration of the immunoreactivity of said antigen in conjunction with the use of known amounts of unlabelled antigen in order that a calibration curve may be constructed.
  • the calibration curve is then used to measure the amount of antigen in samples treated similarly.
  • a competitive displacement assay is described in US 4,895,809, but this assay does not permit the measurement of differential expression of one or more proteins of interest within two unlabelled samples, and without the use of a calibration curve.
  • Disclosed herein is a novel method applicable to arrayed antibodies, other capture agents or interacting biomolecules irrespective in each case of their relative affinities and that permits the reliable assessment of differential protein expression in a complex sample even between different cell types or tissue types without the use of a calibration curve.
  • the method is tolerant towards high levels of non-specific binding and does not require any potentially interaction-disrupting labelling of a test sample, such as but not limited to, a reference or control sample, or a test sample. Most particularly, it enables the comparison of two or more unlabelled samples over a wide concentration range that has hitherto been impractical.
  • a method of comparing the level of at least one biomolecule within a sample with the level of said biomolecule in at least one other sample comprising:
  • the invention provides a method of comparing the level of a target protein within a first sample with the level of said target protein within a second or subsequent sample, the method comprising: (a) contacting a labelled reference sample containing said target protein in the presence of the first sample with an immobilised protein binding unit capable of capturing said target protein;
  • step (b) for each second or subsequent sample contacting a labelled reference sample which is a replicate of that of step (a) in the presence of the second or subsequent sample with a immobilised protein binding unit which is a replicate of that of step (a);
  • step (c) detecting the level of label present on the protein binding units employed in steps (a) and (b) and from a comparison of said levels, determining a comparison of said target protein levels within the first sample and within the second or subsequent sample.
  • the method of the invention employs at least one protein binding unit (PBU) immobilised on any substrate known in the art, such as but not limited to, beads, derivatized glass slides or HydrogelTM pads.
  • the substrate may comprise a bead, a substantially planar surface or any suitable substrate known in the art such as, but not limited to, metal, glass, plastic, HydrogelsTM (Packard BioSciences, Meridian, CT) or membranes, or those documented by Walter et al. in 2000 (Curr. Opin. Microbiol. 3: 298-302).
  • a PBU refers to at least one capture agent. Where a plurality of PBUs are used they are preferably arranged in a specific, co-ordinated pattern e.g. in a grid format, such that the location of each binding unit is known. Such a format is referred to herein as an affinity array.
  • PBUs may be minimal recognition units comprising capture agents which include antibodies, antibody fragments, proteins, polypeptides, peptides, receptors, other biomolecules or complementarity determining regions (CDRs) and even nucleic acids.
  • Capture agents for the production of affinity arrays of PBUs include, but are not limited to, antibodies such as, but not limited to, polyclonal, monoclonal, bispecific, humanized or chimeric antibodies, single chain antibodies, Fab fragments and F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic antibodies, and epitope-binding fragments of any of the above produced by means well-known in the art, as well as but not limited to, proteins, polypeptides, peptides, other biomolecules, aptamers or complementarity determining regions (CDRs).
  • variable heavy and light chains each contribute three regions called CDRs that are responsible for binding antigen.
  • Each region is typically 7-20 amino acids long, e.g. 15-17 amino acids, and its sequence defines the specificity and affinity of that CDR for the antigen.
  • CDRs can be produced by recombinant means or can be chemically synthesized.
  • CDRs can be chemically synthesized according to known CDR sequences using either standard protein synthesis or using a combinatorial synthesis approach.
  • a PBU may be of unknown sequence content, i.e. the identity of the PBU or any bound biomolecule can be determined retrospectively, if desired, by determining the sequence of both using methods known in the art.
  • a target protein includes proteins and glycoproteins, polypeptides and peptides and also includes protein-nucleic acid complexes. Proteins are generally from a biological source such as a cell, tissue or biological fluid.
  • a biological fluid includes a sample obtained from any site (e.g. blood, plasma, serum, bile, urine, cerebrospinal fluid, aqueous or vitreos humor, or any bodily secretion), a transudate, an exudate (e.g.
  • fluid obtained from an abcess or any other site of infection or inflammation or fluid obtained from a joint (e.g. a normal joint or a joint affected by a disease such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), a tissue sample (e.g. a biopsy, blood cells, smears) or homogenates and extracts, including cytoplasm, membranes, and organelles thereof.
  • a tissue sample e.g. a biopsy, blood cells, smears
  • homogenates and extracts including cytoplasm, membranes, and organelles thereof.
  • Cell cultures and culture fluid are also biological samples.
  • a biological sample may be pre-treated as required.
  • Such a treatment may comprise fractionation, differential extraction (to produce e.g. membrane and cytosolic fractions); selective depletion (e.g. for removal of albumin, haptoglobin, immunoglobin G); and application to any specific affinity column (e.g. mannose-6-phosphate receptor for lysosomal enzymes; Sleat and Lobel, 1997, J. Biol. Chem. 272:731-8).
  • the method of the invention overcomes the problems of high levels of background label signals inherent in using proteins and especially labelled proteins, and also overcomes issues associated with non-specific binding by using a competitive assay format where specific protein binding to the immobilised PBUs is distinguished from non-specific binding, or from non-specific label adsorption.
  • binding of a labelled reference sample is measured in the presence of either an unlabelled first sample or a second (or subsequent) unlabelled sample, for example an unlabelled reference or an unlabelled test sample where the reference sample is the first sample and the unlabelled test sample, the second (or subsequent) sample.
  • the unlabelled reference sample may alternatively be the second sample and the unlabelled test sample, the first sample.
  • the unlabelled reference and the unlabelled test samples i.e.
  • an unlabelled first and a second (or subsequent) unlabelled sample can therefore be compared in their ability to compete for binding to the immobilised PBU with the labelled reference sample.
  • the following text refers to a first sample as an unlabelled reference sample and a second or subsequent sample as a test sample.
  • the concentration of the target protein in the test sample may be determined absolutely. If the concentration of target protein contained in the reference sample is not known absolutely then the relative concentration of target protein in the test sample may be determined.
  • the method described herein substantially compensates for, and can totally eliminate background issues and advantageously no calibration is required.
  • a labelled reference sample includes any sample used for displacement by an unlabelled first sample or second (or subsequent) samples using the method of the invention.
  • the labelled reference sample is identical to the unlabelled reference sample (i.e. from the same source with components present in the same amounts), but has been labelled for detection purposes.
  • the labelled reference sample and the unlabelled reference samples are non-identical and can be from different biological sources, e.g. different cells or tissues.
  • samples from different biological sources are from different sources eg different cells or tissues within the same organism.
  • the biological sources are sources from humans.
  • the labelled reference sample can also be a pool of protein samples derived from different biological sources.
  • the disclosed method can employ any labelled reference protein mixture providing said labelled reference sample contains components that bind to all PBUs.
  • This allows the use of generic labelled biomolecular pools, for example but without limitation, obtained by averaging a variety of different cell proteomes (by physical mixing of the protein extracts). This may result in a reduction in the protein concentration range resolved, but significantly, the use of such generic labelled pools would lead to further significant reductions in the time and cost of using arrays of PBUs.
  • test sample includes any unlabelled sample that is to be compared with an unlabelled reference sample. It will be understood by one skilled in the art that test samples may also be compared with each other. Most preferably, test samples are from the same biological source such as the same tissue, cell or biological fluid type as the unlabelled reference samples. Alternatively, they are from different biological sources. In one embodiment, the labelled reference sample is derived from more than one biological source. In a particular embodiment, the labelled reference sample is derived from more than one biological source and the unlabelled reference and test samples are from the same biological source. Preferably, samples are clinical samples from, for example but without limitation, diseased versus normal tissue or cell samples where the normal tissue or cell sample is the reference sample and the diseased tissue or cell sample is the test sample.
  • samples from healthy individuals are compared with samples from diseased individuals and/or with samples from individuals undergoing treatment. It is understood that using the method of the invention permits the comparison of at least two samples on at least two replicate protein binding arrays with no requirement for labelling of said samples for comparison by competing the binding of a labelled reference sample. This is a substantial advantage where the test sample is in short supply, e.g. a biopsy sample. Using unlabelled samples is also particularly advantageous in that this avoids any modification of the proteins present within the sample which can modify or mask interaction sites and which, in turn, may result in the missing of the detection of a potential target of interest.
  • any potential modification of a protein by fluorescent dyes or any other form of labelling will have no deleterious affect on the results as only the displacement of labelled reference proteins by unlabelled proteins present in the reference and test samples are being compared.
  • the displacement data is not affected, i.e. the comparison is between the displacement of the labelled reference by the unlabelled reference sample with the displacement of the labelled reference by the unlabelled test sample.
  • the differential protein composition of two or more samples of interest is assessed such that the displacement of a labelled reference sample e.g. a fluorescently-labelled reference sample, by an identical or similar reference sample that is unlabelled, is compared with the displacement of an identical amount of the same labelled reference sample by an unlabelled test sample.
  • a labelled reference sample e.g. a fluorescently-labelled reference sample
  • an identical or similar reference sample that is unlabelled is compared with the displacement of an identical amount of the same labelled reference sample by an unlabelled test sample.
  • Specific protein binding to immobilised PBUs is distinguished from the non-specific binding (which is automatically compensated for, see formulas below) or label adsorption (which can also be due to labelling of for example, immobilised antibody by the fluorescent dye remaining in the labelled reference sample) using this competitive assay format.
  • the binding of the 1 ⁇ g of a fluorescently-labelled protein present in the labelled sample is competed with an equal amount (1 ⁇ g) of the same protein that is not labelled, the decrease in the recorded fluorescence signal will correspond to approximately 50% of the specific binding values, with the rest of the signal being due to non-specific binding.
  • the displacement values vary slightly due to chemical differences introduced during protein labelling (see Example 2 in the Experimental Section) or if affinity of said binding is low. Displacement using a 100 to 1000-fold excess of unlabelled sample will displace most or all of any specifically bound protein.
  • the method of the invention permits the fully quantitative assessment of differential protein composition between samples.
  • the method is particularly useful for assessing differential expression or post translational modification of proteins, and in diagnosis and screening where differential expression or post translational modification of proteins is an indicator of the diseased state.
  • B, background signal measurement for each individual PBU.
  • MAX maximum signal measurement in the absence of displacement (i.e. signal range).
  • IC 50 concentration of an individual protein in each sample, required for a 50% reduction in a signal (is a constant value dependent upon the respective K D and [L,].
  • [K,] concentration of an individual protein in the unlabelled test sample.
  • [O ⁇ ] concentration of an individual protein in the unlabelled reference sample.
  • Signal signal measured for each target protein in the sample (S ⁇ in the unlabelled test sample; So in the unlabelled reference sample).
  • ] concentration of an individual protein in the labelled reference sample.
  • J_ -J_ Signal K
  • the lack of dependence of the described method on the reagents used in the production of an affinity array (preparation of which is described below) and on the protein composition of the unlabelled reference and test samples means that the same array format can be used with a variety of sample types.
  • the competitive nature of the disclosed method makes it independent of the protein composition of the unlabelled reference and test samples, unlike the direct binding format of typical array applications where a labelled protein or nucleic acid is bound to an immobilised antibody or nucleic acid or vice-versa and in which the binding signal depends not only on sample concentration, but also on the affinity of the PBU and crossreactivity of the affinity reagents used.
  • the displacement achieved by using 1 :1 ratio of labelled:unlabelled reference sample will result in approximately 50% displacement regardless of the affinity of the capture reagents or individual protein concentrations in the sample, as long as sample concentrations are kept high (» respective K D ).
  • the disclosed method can quantitatively characterise protein concentration in an extremely wide dynamic range. The lower range is limited by the sensitivity of the detection technique (similar to a direct binding approach) but the upper range is unlimited.
  • Suitable detection techniques include without limitation, fluorescent scanners (for use with fluorescently labelled probes), phospho-imagers (for detecting radioactively labelled samples), MALDI mass spectrometers for use with isotopically or chemically labelled proteins, surface plasmon resonance-based devices for detecting proteins of different sizes.
  • known signal amplification techniques are used to amplify the differential signals obtained by the described competitive assay.
  • an appropriate "label" should be selected for labelling the reference sample (i.e.
  • more than two affinity arrays (each containing a plurality of PBUs) can be used to assess displacement by more than two samples.
  • a range of unlabelled reference and test sample amounts is used for displacement of the labelled reference sample such that a displacement curve can be prepared for the assessment of the concentration of the displacing unlabelled reference and test samples of interest.
  • Chosen capture agents are preferably immobilized on a single substrate to form an array of PBUs.
  • the array is laid out in a grid form at, for example a grid containing 4 or more, preferably 16 or more PBUs. Selection of the solid support and the methods for immobilization depend on the type of capture agents. Immobilization may be performed through covalent or non-covalent binding.
  • the PBUs are covalently bound to the surface of the substrate via reactive groups or cross-linking agents.
  • PBUs are proteins, such as receptors or antibodies
  • covalent immobilization may be achieved through free cysteines using thiol reactive substrates, or through lysines using amine reactive substrates.
  • heterobifunctional crosslinkers may be used to cross link -NH groups of the substrate to sulfhydryl (-SH) groups of cysteine residues, e.g. BMPS (beta- maleimidopropionic acid N-hydroxysuccinimide ester) can be used to cross link -NH groups to -SH groups on cysteine residues of antibodies.
  • BMPS beta- maleimidopropionic acid N-hydroxysuccinimide ester
  • Non-covalent interactions may be achieved by using avidin- or streptavidin-coated substrates and biotinylated PBUs; protein-A or protein-G coated substrates and Fc containing PBUs (e.g. immunoglobulins); and metal-chelate substrates and histidine tagged PBUs.
  • PBUs are multiple single chain antibodies (SCAs) specific to different peptides. They are purified from phages and solubilised in PBS at similar concentrations (about 0.1 mg/ml). The specific SCAs are placed in multiwell plates (e.g. 96- well) and used to generate a microarray system. It is preferable when dispensing proteins to employ a system that does not use steel pins or contact dispensing equipment. Thus, a preferred system is the Packard BCA Piezo robot (US 5,927,547), which dispenses small volume drops (less than 1 nl) from a glass capillary from above the surface of the microarray substrate.
  • SCAs single chain antibodies
  • a preferred substrate is an aldehyde-activated polyacrylamide pad immobilized on glass or oxidised silicon.
  • a preferred size for the polyacrylamide pad is 2cm x 2cm x 20 ⁇ m (hydrated depth).
  • About 300pl of each SCA is dispensed into a discrete area within the polyacrylamide pad to create a 2-dimensional array in a 3-dimensional structure.
  • the resulting PBU is preferably about 200 ⁇ m in diameter.
  • the free aldehydes within the polyacrylamide react with the amines in the SCAs so that the SCAs are covalently immobilized. Once this reaction is complete (e.g. 30 min after dispensing) any remaining aldehydes can be reduced with sodium borohydride or blocked using reagents containing amino groups (e.g. TRIS buffer) and/or free amino acids.
  • Hydrogel on which PBUs such as antibodies may be immobilized, e.g. via the -NH group of lysine residues to form a Schiff base with aldehyde groups present in the Hydrogel.
  • PBUs such as antibodies
  • the substrate is produced by casting a thin agarose gel on a glass slide, using suitable gaskets or other spacers, and siliconised cover glass or cover slip or any similar solid substrate to produce a gel of uniform thickness. Following casting, the gel is activated with CNBr.
  • agarose gel is preferred over commercial Hydrogel (e.g. from Packard Instrument Co., Meriden, CT) as agarose retains moisture more efficiently and thus allows for spotting of dehydration-sensitive reagents.
  • Another advantage of using thin agarose gel films is that they allow for faster diffusion of compounds due to a larger pore size. Therefore, shorter washing steps may be used. This is in contrast to acrylamide based Hydrogel, which require an increased washing time.
  • a further advantage of 3-dimensional gel-based immobilization techniques is that, through careful choice of physical and physicochemical gel properties, it is possible to allow target peptide fragment- PBU binding to closely approximate the binding likely to occur in free solution i.e. less impeded by solid substrates.
  • single chain Fv antibodies are histidine-tagged.
  • the histidine tag can then be used for immobilization, e.g. using metal chelate affinity on nickel- modified substrates.
  • immobilization examples include using thin membranes (such as nitrocellulose, nylon (charged or uncharged), PVDF and/or their derivatives) attached to or immobilized on glass slides or other solid substrates.
  • thin membranes such as nitrocellulose, nylon (charged or uncharged), PVDF and/or their derivatives
  • attaching commercially available membranes nitrocellulose, supported nitrocellulose, nylon, charged nylon or PVDF membranes from Schleicher & Schuel, UK Ltd., Amersham, Millipore and other suppliers
  • CASTTM layer of Nytran® SuPerCharge positively charged nylon membrane affixed to the glass
  • FASTTM slides microporous polymeric substrate cast onto glass from Schleicher & Schuel, UK, Ltd
  • PBUs may be dispensed using piezo robots similar to the one described above for SCAs.
  • nitrocellulose or nylon based supports have a much higher protein binding capacity compared to Hydrogel or 2 dimensional solid supports (glass, silicon, plastic, etc.).
  • solid support materials that may be used in the microarray are as follows: Binding capacity (highest to lowest): a) immobilized nitrocellulose; immobilized nylon (charged > uncharged) b) FAST; CAST c) Hydrogel d) derivatized solid substrates (glass, silicon, plastics)
  • PBU geometry (best to worst): a) derivatized solid substrates (glass, silicon, plastics) b) FAST slides/CAST slides c) immobilized nitrocellulose; immobilized nylons
  • Moisture retaining (best to worst): a) agarose pads b) acrylamide pads (Hydrogel) c) immobilized membranes or derivatized solid substrates (glass, silicon, plastics)
  • Another preferred immobilization technique uses a combination of non-covalent affinity immobilization coupled to covalent cross-linking.
  • This technique is especially advantageous for immobilizing PBUs such as antibodies, as it allows the antibodies to be attached in an orientation whereby their active sites are easily accessible, and at the same time, remain covalently cross-linked to the solid support.
  • This technique includes, but is not limited to, using Hydrogel (Packard Instrument Co., Meriden, CT) for immobilization of protein-A or protein-G, followed by treatment with glutaraldehyde (high concentration for short time). This results in covalent cross-linking of the protein-A/G to the solid support, and also in aldehyde derivation of solution-exposed amino groups of the protein-AG.
  • Glutaraldehyde pre-treatment of the immobilized Protein-A/G prior to antibody spotting is a preferred technique because it avoids incubating the PBUs (i.e., the antibodies) with the cross-linking agent (i.e. glutaraldehyde), which could result in the derivatization of amino groups of the antibodies.
  • the PBUs i.e., the antibodies
  • the cross-linking agent i.e. glutaraldehyde
  • nucleic acids as PBUs, e.g. as nucleic acid arrays, will allow the capture of proteins containing nucleic acid-binding domains.
  • Nucleic acid arrays may be formatted as "gene chips" and related arrays of oligonucleotides, cDNAs, and other nucleic acids, which are well known in the art (see for example the following: US 6,045,996; 6,040,138; 6,027,880; 6,020,135; 5,968,740; 5,959,098; 5,945,334; 5,885,837; 5,874,219; 5,861 ,242; 5,843,655; 5,837,832; 5,677,195 and 5,593,839.
  • Samples may be labelled according to any standard method known in the art to facilitate detection. Preferred methods include the use of Fluorophore-iodoacetamide, -maleimide or -succinimide groups (e.g. Molecular Probes) Cy3, Cy5 or TAMRA dyes for example via labeling cysteines or lysines (e.g. Alexa Fluor dyes). Another preferred method makes use of NHS ester Bodipy TMR (Molecular Probes, Leiden, Netherlands). Samples may also be labelled via aldehyde or ketone groups (which may be selectively introduced), using hydrazine dyes (Molecular Probes).
  • labels for use in the invention include but are not limited to fluorescein isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially Eu 3+ , chemiluminescent molecules, radio-isotopes ( 125 l, 32 P, 35 S, chelated Tc) or magnetic resonance imaging labels.
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • TR Texas red
  • rhodamine free or chelated lanthanide series salts, especially Eu 3+ , chemiluminescent molecules, radio-isotopes ( 125 l, 32 P, 35 S, chelated Tc) or magnetic resonance imaging labels.
  • Metabolic (or biosynthetic) labeling with [ 35 S]-methionine is also contemplated as well as labeling with [ 14 C]-amino acids and [ 3 H]-a
  • one of a first, second and subsequent samples is a sample from a non-diseased subject, one is a sample from a diseased subject and one is a sample from the test subject.
  • the method will be performed in order to assess if levels of characteristic proteins are raised or lowered, such raising or lowering known to be characteristic of the diseased state, or characteristic of prognosis. The levels can also be monitored in a subject under therapy to assess the response to therapy. Those proteins the raising or lowering of levels of which are characteristic of the diseased state are either or known or may be determined by conventional methods.
  • kits for use in a method which comprises: (a) a labelled reference sample; (b) an unlabelled first sample from a non-diseased subject serving as a negative control;
  • the replicate substrates are used in the steps of the method: one in step (a) for use with the unlabelled first sample and two or more in step (b) for use with the second sample and the sample(s) from the test subject(s). Differential binding of the samples to the replicate substrates is capable of indicating relative levels of characteristic proteins as discussed earlier in the description.
  • diagnostic kit for use in a method of diagnosing disease, which disease is characterised by a raised or lowered level of characteristic target proteins in a sample from the test subject.
  • the method of the invention has significant advantages over current technologies, viz: 1. labelling of the samples for comparison is not required.
  • the invention permits the comparison of unlabelled samples, thus avoiding inaccuracies due to sample modifications produced by the introduction of a label.
  • the use of non-labelled samples means that no purification step is required resulting in faster and cheaper experiments.
  • generic arrays, protocols and detection means can be used for any sample type, regardless of its protein composition, thus enabling mass production that is standard and cost effective.
  • the method is especially suitable for protein samples that are in limited supply such as, but without limitation, biopsy samples.
  • a single labelled reference sample e.g. an appropriate control or untreated sample, can be prepared on a large scale for use in all experiments, with different unlabelled test samples used for displacement.
  • the method also allows the use of a generic protein mixture for labelling thus further reducing the cost and time spend for each experiment.
  • 6. a broad dynamic range of protein concentrations is resolvable with no upper limit.
  • the method does not depend on the affinity of the capture reagents used.
  • the method is equally applicable to a variety of assays such as but not limited to, protein- protein interactions, antibody-protein interactions, and protein-nucleic acid interaction pairs.
  • Figure 1 is a map showing the positions of substrate-immobilised PBUs comprising capture agents in an affinity array of 74 PBUs.
  • Figure 2 is a scanned image of five arrays labelled l-V, showing the degree of fluorescent labelling of both immobilised antibodies and of the background acrylamide gel obtained using five fluorescent dyes (see Example 1 a).
  • Figure 3 is a series of histograms showing the degree of fluorescent labelling of the immobilised antibodies (black fill) and of the background acrylamide gel (no fill) obtained using five fluorescent dyes (see Example 1 a).
  • the histogram stacks labelled l-V correspond to those in Figure 2.
  • Figure 4 is a series of histograms showing the displacement of fluorescently-labelled reference whole liver lysate by various amounts (indicated in panel D) of an identical unlabelled reference whole liver lysate on an array comprising PBUs (see Example 1 b).
  • Panel A relates to row A, columns 1 -19 of Figure 1 ;
  • Panel B relates to row B, columns 1 -19 of figure 1 ;
  • Panel C relates to row C, columns 1 -19 of Figure 1 ;
  • Panel D relates to row D, columns 1-19 of Figure 1.
  • Figure 5 shows a series of histograms of the results obtained from an array of substrate- immobilised PBUs comprising 4x18 antibodies contacted with labelled ovary lysate in the presence of unlabelled ovary lysate (unlabelled reference sample) or unlabelled kidney lysate (test sample) as described in Example 2. Filled bars represent the difference between the unlabelled reference sample and unlabelled test sample (ovary sample minus kidney sample).
  • Each panel (A-D) corresponds to a single row of PBUs (see Table 1). Open bars in each case show mean differences obtained by averaging data from four PBUs (each PBU was printed in quadruplicate). Filled bars show mean differences obtained by averaging data from two PBUs (i.e. two outlying values were disregarded in each case).
  • Figure 6 is a repetition of the experiment described in Figure 5 except that the test sample (kidney lysate) was used in x10 excess, i.e a 1 :10 ratio (by total protein) compared to the labelled reference sample, whilst the unlabelled reference sample (ovary lysate) was used at 1 :1 ratio with the labelled reference sample (see Example 3).
  • Panels A-D and open and filled bars are as in Figure 5.
  • PBUs were prepared by dispensing capture agents using an arrayer (BioChip Arrayer with Piezo-tips, Packard Bioscience, Meriden, CT). To prepare PBUs, slides were incubated with the dispensed capture agent in a sealed, humidified chamber overnight.
  • Tris-Buffered Saline 0.05M Tris, 0.15M NaCI, pH7.6:glycine (1.5% w/v in water):bovine serum albumin (1% w/v final), followed by a final rinse in water before drying.
  • the slides comprising the PBUs were then ready for use.
  • the fluorescent dyes detailed below were prepared (at equivalent concentrations) in 1 x Tris-Buffered Saline plus bovine serum albumin (1 % w/v final). Each dye was incubated with one of five identical arrays (l-V) prepared as described above at room temperature for 1 hr. Arrays were washed in H 2 O for 15min with several changes of water. The identities and positions of each PBU are shown in Figure 1 (antibodies -columns 1-10, from Sigma, Chemical Co. USA, and transcription factors -columns 11-19; Abeam, Ltd, Cambridge, UK).
  • Fluorescent dyes 1. lodoacetamide-Cyanine ⁇ (-Cy5) - Array I
  • Protein lysates (1mg) were labelled using an excess concentration of iodoacetamide cyanine-5 dye in phosphate buffered saline buffer, pH7.4, plus Tween (0.5% v/v) at room temperature for 3hr. Following labelling, protein lysates were purified from unconjugated dye by gel-filtration using p-6 chromatography columns (BioRad). Samples were centrifuged at 1 ,000 x g for 5min to isolate labelled proteins from free, unconjugated dye (samples passed through 2 separate columns).
  • Fluorescence measured after hybridisation in the absence (O ⁇ g) of unlabelled protein includes both nonspecific binding/adsorption and specific binding components and is maximal, whereas fluorescence measured after hybridisation of 1 ⁇ g of labelled reference whole liver lysate in the presence of 10O ⁇ g of unlabelled whole liver lysate contains only non-specific binding/adsorption (and is minimal) and was subtracted from all the experimental data.
  • Hydrogel glass slides (Packard Bioscience, Meriden, Connecticut, USA) were soaked overnight in 50% glutaraldehyde, washed with H 2 O and air-dried.
  • the antibodies were dispensed onto the Hydrogel slides (acrylamide-based pads) using a BioChip Arrayer with Piezo-electric tips (Packard Bioscience, Meriden, Connecticut, USA). A total of 72 antibodies were arrayed in quadruplicate onto each slide (see Table 1).
  • the binding of fluorescent dye molecules onto the slides was measured using a Biochip Imager Confocal laser scanning system at the highest resolution of 10 ⁇ m.
  • Total fluorescence signals corresponding to each PBU obtained by hybridisation in the presence of various displacing samples) were recorded. Mean values (obtained by averaging the quadruplicate readings for each antibody) were used for analysis.
  • the set of values corresponding to the hybridisation in the presence of unlabelled kidney sample was subtracted from an analogous set obtained when unlabelled ovary was used. When the subtraction results in a positive value, this indicates that the signal measured for the unlabelled ovary sample is stronger, i.e. less labelled ovary displaced; thus a higher concentration of the corresponding protein exists in the kidney sample.
  • a negative value means lower protein expression in the kidney sample.
  • Figure 5 shows displacement data obtained for an array of 72 PBUs, incubated and bound with 1 ⁇ g labelled ovary lysate and displaced by either 10 ⁇ g unlabelled ovary lysate or by 10 ⁇ g unlabelled kidney lysate.
  • the plots ( Figures 5A-D) show the differences (ovary displacement minus kidney displacement) obtained at approximately 90% overall displacement level (e.g. 10-fold excess of the unlabelled sample, by total protein concentration). Normalised data were obtained as described previously. In all cases, at least one array is required for each test sample.
  • the displacement protocols described herein result in reliable and meaningful data, unlike a direct (non-competitive) hybridisation approach, which works best with nucleic acids.
  • the results shown in Figure 5 indicate a number of differentially expressed proteins.
  • the highest differential expression was detected using anti-HLA-class I antigen antibody, anti-blood-brain barrier (neurothelin) HT-7 antibody, anti-MAD (helix loop helix - leucine/zipper transcription factor) antibody, anti-phosphothreonine antibody, anti-l ⁇ B alpha antibody and anti-TATA binding protein antibody.
  • the results are in a good agreement with data reported previously. For example, reports by Le, et al. (2002, Exp. Mol. Med. 34:18-26) and Dolo, et al. (1997, Oncol Res 9:129-38) indicate expression of HLA-1 in ovary reaching especially high levels in ovarian tumours.
  • the negative value of the HLA-1 ( Figure 5A, no.4) is indicative of higher HLA-1 content in the ovary sample.
  • the blood brain barrier protein, neurothelin, recognised by the HT-7 antibody is predominantly expressed by brain endothelial cells. It has also been reported to be expressed in basolateral membranes of kidney tubules (Risau, W. et al., 1986, EMBO J. 5:3179-8313). As such, a higher level of this protein was found in kidney lysate compared to ovary ( Figure 5A, no.6).
  • Anti- phosphothreonine antibody signal (Figure 5C, no.6) is indicative of a higher level of phosphorylated proteins in kidney.
  • TATA-binding protein is a transcription factor, which plays an important role in eukaryotic gene expression and is present in both ovary and kidney on mRNA level (UniGene) shows a higher level of expression in kidney ( Figure 5C, peak 15).
  • Example 3 This example demonstrates the tolerance of the method of the invention to variability in the protein concentration of unlabelled reference and test samples.
  • the same experiment as described in Example 2 was performed except that an array of 72 PBUs was contacted with 1 ⁇ g of labelled ovary lysate in the presence of either 1 ⁇ g unlabelled ovary lysate or by 10 ⁇ g unlabelled kidney lysate (see Figure 6).
  • a complete set of subtracted values was averaged and the obtained average value was subtracted from each individual peak.
  • Figure 6 demonstrates substantially similar results to those shown in Figure 5, with differential expression detected using anti-HLA-class I antigen antibody, anti-blood-brain barrier (neurothelin) HT-7 antibody, anti-MAD (helix loop helix - leucine/zipper transcription factor) antibody, anti-phosphothreonine antibody, anti-l ⁇ B alpha antibody and anti-TATA binding protein antibody.
  • HLA-1 Figure 6A, no. 4
  • MAD protein (figure 6A, no.

Landscapes

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

Abstract

L'invention concerne un nouveau procédé de quantification de niveaux différentiels de protéines au moyen d'échantillons non marqués, en particulier au moyen de réseaux à liaison protéinique. Ce procédé sert à évaluer une expression différentielle ou une modification post-translationnelle des protéines, ainsi qu'à diagnostiquer et à cribler l'expression différentielle ou la modification post-translationnelle des protéines lorsque celles-ci se révèlent être un indicateur de l'état pathologique.
PCT/GB2003/000112 2002-01-11 2003-01-13 Procede de quantification de niveaux de proteines dans des echantillons biologiques WO2003058249A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003201657A AU2003201657A1 (en) 2002-01-11 2003-01-13 Method for quantitation of protein levels in biological samples

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0200595.7 2002-01-11
GB0200595A GB0200595D0 (en) 2002-01-11 2002-01-11 Quantitation of differential expression

Publications (1)

Publication Number Publication Date
WO2003058249A1 true WO2003058249A1 (fr) 2003-07-17

Family

ID=9928930

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2003/000112 WO2003058249A1 (fr) 2002-01-11 2003-01-13 Procede de quantification de niveaux de proteines dans des echantillons biologiques

Country Status (3)

Country Link
AU (1) AU2003201657A1 (fr)
GB (1) GB0200595D0 (fr)
WO (1) WO2003058249A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2388371A (en) * 2002-03-25 2003-11-12 Sense Proteomic Ltd Protein arrays
US7460960B2 (en) 2002-05-10 2008-12-02 Epitome Biosystems, Inc. Proteome epitope tags and methods of use thereof in protein modification analysis
US7618788B2 (en) 2002-05-10 2009-11-17 Millipore Corporation Proteome epitope tags and methods of use thereof in protein modification analysis
US7645586B2 (en) 2006-03-23 2010-01-12 Millipore Corporation Protein isoform discrimination and quantitative measurements thereof
US7867755B2 (en) 2000-10-31 2011-01-11 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen Method for analyzing proteins

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4895809A (en) * 1984-01-09 1990-01-23 Varian Associates, Inc. Immobilized antigen-antibody displacement process
WO1998024935A1 (fr) * 1996-12-06 1998-06-11 Urocor, Inc. DIAGNOSTIC D'ETAT PATHOLOGIQUE A L'AIDE DE PROFILS d'ARN MESSAGER
US6197599B1 (en) * 1998-07-30 2001-03-06 Guorong Chin Method to detect proteins
WO2002010338A2 (fr) * 2000-07-31 2002-02-07 Gene Logic, Inc. Expression d'une proteine du type gage/page dans l'hypertrophie benigne de la prostate

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4895809A (en) * 1984-01-09 1990-01-23 Varian Associates, Inc. Immobilized antigen-antibody displacement process
WO1998024935A1 (fr) * 1996-12-06 1998-06-11 Urocor, Inc. DIAGNOSTIC D'ETAT PATHOLOGIQUE A L'AIDE DE PROFILS d'ARN MESSAGER
US6197599B1 (en) * 1998-07-30 2001-03-06 Guorong Chin Method to detect proteins
WO2002010338A2 (fr) * 2000-07-31 2002-02-07 Gene Logic, Inc. Expression d'une proteine du type gage/page dans l'hypertrophie benigne de la prostate

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7867755B2 (en) 2000-10-31 2011-01-11 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen Method for analyzing proteins
US8241894B2 (en) 2000-10-31 2012-08-14 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen Method for analyzing proteins
GB2388371A (en) * 2002-03-25 2003-11-12 Sense Proteomic Ltd Protein arrays
US7460960B2 (en) 2002-05-10 2008-12-02 Epitome Biosystems, Inc. Proteome epitope tags and methods of use thereof in protein modification analysis
US7618788B2 (en) 2002-05-10 2009-11-17 Millipore Corporation Proteome epitope tags and methods of use thereof in protein modification analysis
US7964362B2 (en) 2002-05-10 2011-06-21 Millipore Corporation Proteome epitope tags and methods of use thereof in protein modification analysis
US8244484B2 (en) 2002-05-10 2012-08-14 Emd Millipore Corporation Proteome epitope tags and methods of use thereof in protein modification analysis
US7645586B2 (en) 2006-03-23 2010-01-12 Millipore Corporation Protein isoform discrimination and quantitative measurements thereof
US7855057B2 (en) 2006-03-23 2010-12-21 Millipore Corporation Protein splice variant/isoform discrimination and quantitative measurements thereof

Also Published As

Publication number Publication date
AU2003201657A1 (en) 2003-07-24
GB0200595D0 (en) 2002-02-27

Similar Documents

Publication Publication Date Title
Kusnezow et al. Antibody microarrays: promises and problems
Cretich et al. Protein and peptide arrays: recent trends and new directions
Pavlickova et al. Advances in recombinant antibody microarrays
US8906832B2 (en) Quantitative analysis of carbohydrate-protein interactions using glycan microarrays: determination of surface and solution dissociation constants
CN100420947C (zh) 用单一捕获剂定量检测特异性分析物的方法及其试剂盒
US20110306511A1 (en) Methods for multiplex analyte detection and quantification
US20070054326A1 (en) Antibody-based protein array system
US6921637B2 (en) Colloid compositions for solid phase biomolecular analytical, preparative and identification systems
US20090017455A1 (en) Methods and systems for detecting and/or sorting targets
JPH10319011A (ja) 複数の分析物の同時検出デバイス及び装置
Pavlickova et al. Microarray of recombinant antibodies using a streptavidin sensor surface self-assembled onto a gold layer
US20090215073A1 (en) Layered peptide/antigen arrays - for high-throughput antibody screening of clinical samples
Seurynck-Servoss et al. Surface chemistries for antibody microarrays
JP2005528612A (ja) 生体分子の相互作用をモニターするための新規方法
Spisak et al. Biomedical applications of protein microarrays
WO2003058249A1 (fr) Procede de quantification de niveaux de proteines dans des echantillons biologiques
WO2006071132A1 (fr) Micropuce a adn destinee au dosage immunologique parallele multiple de composes, et procedes de dosage immunologique faisant appel a ladite micropuce
US20070037174A1 (en) Chemiluminescent generated fluorescent labeling
WO2002073180A1 (fr) Systeme de microtitrations de proteines base sur des anticorps
US20180045734A1 (en) Chemically functionalized array to analyze protein modifications
Cretich et al. Peptide microarrays on coated silicon slides for highly sensitive antibody detection
US20030224459A1 (en) Protein detection method
Sakanyan et al. 10 Near-Infrared Fluorescence Detection of Antigen–Antibody Interactions on Microarrays
Beusink Label-free biomolecular interaction sensing on microarrays using surface plasmon resonance imaging
JPH10506996A (ja) 多種検体の同時分析用結合マトリックスならびに分析要素

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP