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WO2006119886A1 - Collagene de type iv utilise comme cible/marqueur pour la resistance a l'insuline - Google Patents

Collagene de type iv utilise comme cible/marqueur pour la resistance a l'insuline Download PDF

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Publication number
WO2006119886A1
WO2006119886A1 PCT/EP2006/004066 EP2006004066W WO2006119886A1 WO 2006119886 A1 WO2006119886 A1 WO 2006119886A1 EP 2006004066 W EP2006004066 W EP 2006004066W WO 2006119886 A1 WO2006119886 A1 WO 2006119886A1
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Prior art keywords
collagen type
insulin resistance
compound
protein
diabetes
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Inventor
Peter Berndt
Stefan Evers
Michael Fountoulakis
Mitchell Lee Martin
Elena Sebokova
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F Hoffmann La Roche AG
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F Hoffmann La Roche AG
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    • 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
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • 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
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Collagen Type IV as target/marker for insulin resistance
  • Type 2 diabetes is a disease of fast growing worldwide importance and can be described as a failure of the pancreatic beta-cell (beta-cell failure) to compensate, with enhanced insulin secretion of the beta-cells, for peripheral insulin resistance.
  • Insulin resistance can be considered the first step in the development of Type 2 Diabetes and develops years before diabetes is diagnosed. During this first stage, patients remain normo-glycaemic and compensate for reduced insulin responsiveness of muscle and liver by an enhanced secretion of insulin. At later stages in the development of Type 2 Diabetes, beta cell function decreases, leading to impaired glucose tolerance and, finally, diabetes. Early intervention by either weight loss, exercise, or pharmaceutical treatment, was shown to delay or even prevent the development of diabetes in patients with impaired glucose tolerance (Diabetes Prevention Program Research Group, N. Engl. J. Med. 346 (2002) 393-403). Therefore, an early diagnosis of insulin resistance would allow early intervention by anti-diabetic treatment or other measures that would prevent progression of the disease.
  • EHC euglycemic-hyperinsulinemic clamp
  • the aim of the present invention is to identify and provide a novel target to screen for compounds that prevent, attenuate, or inhibit Insulin Resistance, and for a marker that allows for monitoring and/or diagnosis of Insulin Resistance at an earlier stage of type II diabetes and more reliably than can presently be done.
  • Collagen Type IV forms part of basement membranes and is released during their turnover.
  • An increase in plasma Collagen Type IV has been shown previously (Lee et al., J. Korean Med. Sci. 1994, 9, p. 341-346).
  • Collagen Type IV maybe used as a marker for diabetic microalbuminuria (Nicoloff et al., 2001, Pediatr. Diabetes, 2, p. 184- 190 ; Hayashi et al., 1992, Diabet. Med. 9, p. 366-370)
  • the present invention provides a target for the treatment and/or prevention of Insulin Resistance, and a novel marker for the early diagnosis of Insulin Resistance in diabetes.
  • said changes are an increase in the levels of secreted Collagen Type IV.
  • said changes maybe a decrease in the levels of type IV collagen, depending on the stage of disease.
  • novel target and/or marker Collagen Type IV may be used for diagnostic, monitoring as well as for screening purposes.
  • the diagnostic method according to the present invention may help to assess efficacy of treatment and recurrence of Insulin Resistance in the follow-up of patients. Therefore, the present invention provides the use of protein Collagen Type IV for monitoring the efficacy of treatment of diabetes.
  • the diagnostic method according to the present invention is used for patient screening purposes. I.e., it is used to assess subjects without a prior diagnosis of diabetes by measuring the level of Collagen Type IV and correlating the level of Collagen Type IV to the presence or absence of Insulin Resistance.
  • the methods of the present invention are useful for monitoring progression of the disease through the different stages leading to diabetes, namely Insulin Resistance, Impaired Glucose Tolerance and Diabetes.
  • the present invention thus provides a method for monitoring the progression of diabetes, comprising the steps of (a) providing a liquid sample obtained from an individual, (b) contacting said sample with a specific binding agent for Collagen Type IV under conditions appropriate for formation of a complex between said binding agent and Collagen Type IV, and (c) correlating the amount of complex formed in (b) to the amount of complex formed in Insulin Resistance.
  • the present invention also provides a method for monitoring the efficacy of treatment of diabetes, comprising the steps of (a) providing a liquid sample obtained from a patient treated against diabetes, (b) contacting said sample with a specific binding agent for Collagen Type IV under conditions appropriate for formation of a complex between said binding agent and Collagen Type IV, and (c) correlating the amount of complex formed in (b) to the amount of complex formed in the absence of treatment.
  • the present invention provides an in vitro method of screening for a compound which interacts with Collagen Type IV, comprising the steps of a) contacting protein
  • Collagen Type IV with a compound or a plurality of compounds under conditions which allow interaction of said compound or a plurality of compounds with Collagen Type IV; and b) detecting the interaction between said compound or plurality of compounds with said polypeptide.
  • in vitro method as used herein relates to methods performed with cell cultures or cell-free methods, but not with whole organisms.
  • the present invention provides an in vitro method of screening for a compound that prevents and/or inhibits and/or attenuates Insulin Resistance, comprising the steps of a) contacting a compound with protein Collagen Type IV; and b) measuring the activity of protein Collagen Type IV; wherein a compound which inhibits or stimulates the activity of protein Collagen Type IV is a compound that may prevent and/or inhibit and/or attenuate Insulin Resistance.
  • said method additionally comprises the step of immobilizing protein Collagen Type IV prior to step a) or between steps a) and b).
  • ,activity as used herein relates e.g. to the ability of Collagen Type IV to mediate migration of cells, or to interact with its receptors, eg with integrins alpha 1 beta 1 and alpha2 betal. Assays to determine the activity of Collagen Type IV are well known in the art.
  • the in vitro screening methods hereinbefore described are cell-free assays.
  • Such assays involve contacting a form of Collagen Type IV (e.g., full-length polypeptide, a biologically active fragment of said polypeptide, or a fusion protein comprising all or a portion of said polypeptide) with a test compound and determining the ability of the test compound to bind to said polypeptide. Binding of the test compound to said polypeptide can be determined either directly or indirectly as described above.
  • the assay includes contacting the said polypeptide with a known compound which binds said polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with said polypeptide, wherein determining the ability of the test compound to interact with said polypeptide comprises determining the ability of the test compound to preferentially bind to the said polypeptide as compared to the known compound.
  • the cell-free assays of the present invention are amenable to use of either a membrane-bound form of a polypeptide or a soluble fragment thereof.
  • a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution.
  • solubilizing agents include non- ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton X-100, Triton X- 114, Thesit, Isotridecypoly( ethylene glycol ether)n, 3-[(3- cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylamminio]-2-hydroxy-l-propane sulfonate (CHAPSO), or N- dodecyl-N, N-dimethyl-3-ammonio-l -propane sulfonate.
  • non- ionic detergents such as n-octy
  • binding of a test compound to a polypeptide, or interaction of a polypeptide with a binding molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione- S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed binding protein or polypeptide, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of binding or activity of a polypeptide hereinbefore described can be determined using standard techniques.
  • a polypeptide hereinbefore described or its binding molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with a polypeptide or binding molecules can be derivatized to the wells of the plate. Unbound binding protein or polypeptide of the invention are trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with a polypeptide hereinbefore described or binding molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with a polypeptide or binding molecule.
  • the present invention also provides a method of screening for a compound that prevents and/or inhibits and/or delays Insulin Resistance, comprising the step of detecting soluble Collagen Type IV secreted from a host in the presence or absence of said compound, wherein a compound that prevents and/or inhibits and/or delays Insulin Resistance is a compound with which the level of Collagen Type IV secreted from a host is changed.
  • a host may be a model cell representing beta-cells in culture, or an animal which can be used as a model for Insulin Resistance.
  • the present invention also provides for a use of protein Collagen Type IV as a target and/or as a marker for screening for a compound that prevents and/or inhibits Insulin Resistance.
  • the diagnostic, monitoring or patient screening methods according to the present invention are based on a liquid sample which is derived from an individual. Unlike methods known from the art Collagen Type IV is specifically measured from this liquid sample by use of a specific binding agent.
  • a specific binding agent is, e.g., a receptor for Collagen Type IV or an antibody to Collagen Type IV.
  • a receptor for Collagen Type IV or an antibody to Collagen Type IV.
  • specific is used to indicate that other biomolecules present in the sample do not significantly bind to the binding agent specific for Collagen Type IV. A level of less than 5% cross-reactivity is considered not significant.
  • a specific binding agent preferably is an antibody reactive with Collagen Type IV.
  • the term antibody refers to a polyclonal antibody, a monoclonal antibody, fragments of such antibodies, as well as to genetic constructs comprising the binding domain of an antibody.
  • Antibodies are generated by state of the art procedures, e.g., as described in Tijssen (Tijssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam).
  • polyclonal antibodies raised in rabbits have been used.
  • polyclonal antibodies from different species e.g. rats or guinea pigs, as well as monoclonal antibodies can also be used. Since monoclonal antibodies can be produced in any amount required with constant properties, they represent ideal tools in development of an assay for clinical routine.
  • the generation and use of monoclonal antibodies to Collagen Type IV in a method according to the present invention is yet another preferred embodiment.
  • Collagen Type IV has been identified as a marker which is useful in the diagnosis of Insulin Resistance
  • alternative ways may be used to reach a result comparable to the achievements of the present invention.
  • alternative strategies to generate antibodies may be used.
  • Such strategies comprise amongst others the use of synthetic peptides, representing an epitope of Collagen Type IV for immunization.
  • DNA immunization also known as DNA vaccination may be used.
  • the liquid sample obtained from an individual is contacted with the specific binding agent for Collagen Type IV under conditions appropriate for formation of a binding agent Collagen Type FV-complex.
  • Such conditions need not be specified, since the skilled artisan without any inventive effort can easily identify such appropriate incubation conditions.
  • the amount of complex is measured and correlated to the diagnosis of Insulin Resistance or to a respective control, as hereinbefore described.
  • the skilled artisan will appreciate there are numerous methods to measure the amount of the specific binding agent Collagen Type FV-complex all described in detail in relevant textbooks (cf., e.g., Tijssen P., supra, or Diamandis, et al., eds. (1996) Immunoassay, Academic Press, Boston).
  • Collagen Type IV is detected in a sandwich type assay format.
  • a first specific binding agent is used to capture Collagen Type IV on the one side and a second specific binding agent, which is labeled to be directly or indirectly detectable, is used on the other side.
  • Collagen Type IV can be measured from a liquid sample obtained from an individual sample. No tissue and no biopsy sample is required to apply the marker Collagen Type IV in the diagnosis of Insulin Resistance.
  • the method according to the present invention is practiced with serum as liquid sample material.
  • the method according to the present invention is practiced with plasma as liquid sample material.
  • the method according to the present invention is practiced with whole blood as liquid sample material.
  • whole blood as liquid sample material.
  • the inventors of the present invention have surprisingly been able to detect protein Collagen Type IV in a bodily fluid sample. Even more surprising they have been able to demonstrate that the presence of Collagen Type IV in such liquid sample obtained from an individual can be correlated to the diagnosis of Insulin Resistance.
  • Antibodies to Collagen Type IV with great advantage can be used in established procedures, e.g., to Insulin Resistance in situ, in biopsies, or in immunohistological procedures.
  • an antibody to Collagen Type IV is used in a qualitative (Collagen Type IV present or absent) or quantitative (Collagen Type IV amount is determined) immunoassay.
  • the present invention relates to use of protein Collagen Type IV as a marker molecule in the diagnosis of Insulin Resistance from a liquid sample obtained from an individual.
  • marker molecule is used to indicate that changes in the level of the analyte Collagen Type IV as measured from a bodily fluid of an individual marks the presence of Insulin Resistance.
  • the use of protein Collagen Type IV itself represents a significant progress to the challenging field of Insulin Resistance diagnosis. Combining measurements of Collagen Type IV with other known markers for diabetes, like insulin, or with other markers of Insulin Resistance yet to be discovered, leads to further improvements. Therefore in a further preferred embodiment the present invention relates to the use of Collagen Type IV as a marker molecule for diabetes, preferably for Insulin Resistance, in combination with another marker molecule for diabetes, preferably for Insulin Resistance, in the diagnosis of diabetes, preferably of Insulin Resistance from a liquid sample obtained from an individual.
  • Preferred selected other diabetes markers with which the measurement of Insulin Resistance may be combined are insulin, pre-insulin, and/or C-peptide.
  • kits which comprises the specific binding agent and the auxiliary reagents required to perform the assay.
  • the present invention therefore also relates to an immunological kit comprising at least one specific binding agent for CollagenType IV, recombinant Collagen Type IV as a standard antigen, binding buffer and the reagents required to detect bound Collagen Type IV.
  • said kit comprises a first specific binding agent for Collagen Type FV, recombinant Collagen Type IV as antigen standard, a second specific binding agent which is coupled to a detection system, and detection reagents, for measurement of Collagen Type IV.
  • said first and second binding reagents are antibodies which recognize different epitopes on Collagen Type IV and which preferably do not crossreact upon detection.
  • Said detection system may be an enzyme bound to the second specific binding reagent, such as, as a non-limiting example, horseradish peroxidase, or in another embodiment, said second specific binding agent is covalently linked to biotin, and the signal is detected with enzyme-labeled avidin/streptavidin.
  • the detection system is not directly coupled to the second specific binding agent, but to a third binding agent, such as an antibody, which specifically bind the second specific binding agent.
  • One way of assessing clinical utility of the novel marker Collagen Type IV is by measuring its levels in 17 patients that were diagnosed as being insulin resistant by measuring the glucose disposal rate with the EHC method and comparing the levels with those measured in 17 patients with demonstrated normal glucose disposal rate as determined by the same methodology. For statistical analysis, standard Student's t-test evaluation is performed with values ⁇ 0.05 being taken as significant.
  • the ROC graph is a plot of all of the sensitivity/specificity pairs resulting from continuously varying the decision threshold over the entire range of data observed.
  • the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease.
  • the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds.
  • sensitivity or the true-positive fraction [defined as (number of true- positive test results) (number of true-positive + number of false-negative test results)]. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • false-positive fraction or 1 - specificity [defined as (number of false-positive results)/(number of true-negative + number of false-positive results) ] . It is an index of specificity and is calculated entirely from the unaffected subgroup.
  • the ROC plot is independent of the prevalence of disease in the sample.
  • Each point on the ROC plot represents a sensitivity/-specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true- positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity).
  • the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
  • Plasma is a difficult to analyse by Proteomics techniques because it includes ca. ten high-abundance proteins, which represent approximately 98% of the total protein mass.
  • the scheme described comprises three chromatography steps, matrix blue, protein G and ion exchange, and is highly reproducible. All chromatographic steps were performed on an FPLC System (Pharmacia).
  • Plasma Human plasma was received from three control individuals and three patients with diabetes type II. Protease inhibitors cocktail (Roche Diagnostics, Mannheim, Germany) was added to the plasma (one tablet to 50 ml). Plasma was diluted three- fold with 25 mM MES, pH 6.0, to reduce the salt concentration and adjust the pH to about 6.0. The two columns, Mimetic blue SA P6XL (50 ml, ProMetic BioSciences Ltd.) and HiTrap Protein G HP (5 ml, Amersham Biosciences) were connected in series and equilibrated with 25 mM MES, pH 6.0.
  • the volume corresponding to approximately one g of plasma protein(15 ml, 66 mg/ml) was filtered through a 0.22 ⁇ m filter and applied onto the Mimetic blue column at 5 ml/min.
  • the flow through of this column was directly loaded onto the Protein G column and the flow-through fraction from the latter column was collected (about 120 mg).
  • the two columns were washed with 100 ml of 25 mM MES, pH 6.0 and then they were separated.
  • the Mimetic blue column was eluted with a step gradient of 2 M NaCl in 50 mM Tris-HCl, pH 7.5 and the Protein G was eluted with 100 mM glycine-HCl, pH 2.8 and the eluate was neutralized with 1 M Tris base.
  • the flow through fraction and the two eluates were analyzed by two-dimensional gels and the proteins were identified by MALDI-MS.
  • the proteins were identified by MALDI-MS.
  • mainly full- length and fragmented albumin were detected.
  • In the eluate from the Protein G column mainly heavy and light Ig chains were detected. Most of the other plasma proteins were recovered in the flow through fraction.
  • Immobilized pH gradient (IPG) strips were purchased from Amersham Biosciences (Uppsala, Sweden).
  • Acrylamide was obtained from Biosolve (Valkenswaard, The Netherlands) and the other reagents for the polyacrylamide gel preparation were from Bio-Rad Laboratories (Hercules, CA, USA).
  • CHAPS was from Roche Diagnostics (Mannheim, Germany), urea from Applichem (Darmstadt, Germany), thiourea from Fluka (Buchs, Switzerland) and dithioerythritol from Merck (Darmstadt).
  • Samples of 0.5 mg total protein were applied on 3-10 NL IPG strips, in sample cups at their basic and acidic ends. Focusing started at 200 V, and the voltage was gradually increased to 5000 V at 3 V/min, using a computer-controlled power supply and was kept constant for a further 6 h. The second-dimensional separation was performed either on 12% constant SDS polyacrylamide gels (180x200x1.5 mm) at 40 mA per gel. After protein fixation for 12 h in 40% methanol that contained 5% phosphoric acid, the gels were stained with colloidal Coomassie blue (Novex, San Diego, CA, USA) for 24 h.
  • colloidal Coomassie blue Novex, San Diego, CA, USA
  • MALDI- TOF-MS Matrix- assisted laser desorption/ionization time-of-flight mass spectroscopy
  • the sample application was performed with a CyBi-WeIl apparatus. Samples were analyzed in a time-of- flight mass spectrometer (Ultraflex TOF-TOF, Bruker Daltonics) in the reflectron mode. An accelerating voltage of 20 kV was used. Proteins were identified on the basis of peptide-mass matching.
  • Mass spectrometric data is two times filtered using a low-pass median parametric spline filter in order to determine the instrument baseline.
  • the smoothed residual mean standard deviation from the baseline is used as an estimate of the instrument noise level in the data.
  • the data point with the largest deviation from the baseline is used to seed a non-linear (Levenberg-Marquardt) data fitting procedure to detect possible peptide peaks.
  • the fit procedure attempts to produce the best fitting average theoretical peptide isotope distribution parameterized by peak height, resolution, and monoisotopic mass. The convergence to a significant fit is determined in the usual way by tracking sigma values.
  • an estimate for the errors of the determined parameters is produced using a bootstrap procedure using sixteen repeats with a random exchange of 1/3 of the data points.
  • the resulting fit is subtracted from the data, the noise level in the vicinity of the fit is adjusted to the sum of the extrapolated noise level and the deviation from the peak fit, and the process is iterated to find the next peak as long as a candidate peak more than five times over level of noise can be found.
  • the process is stopped when more than 50 data peaks have been found.
  • the zero and first order of the time-of flight to mass conversion are corrected using linear extrapolation from detected internal standard peaks, and confidence intervals for the monoisotopic mass values are estimated form the mass accuracies of the peaks and standards.
  • Probabilistic matching of spectra peaks to in-silico protein digests Peak mass lists for mass spectra are directly compared to theoretical digests for whole protein sequence databases. For each theoretical digest, [1-11(1- N P(pi))] cMatches is calculated, where N is the number of peptides in the theoretical digest, P(pi) is the number of peptides that match the confidence interval for the monoisotopic mass of the peak divided by the count of all peptides in the sequence database, and cMatches is the number of matches between digest and mass spectrum. It can be shown that this value is proportional to the probability of obtaining a false positive match between digest and spectrum. Probability values are further filtered for high significance of the spectra peaks that produce the matches.
  • VECs Human Vascular Endothelial Cells
  • RNA samples 10 ⁇ g of total cellular RNA were reverse transcribed (Invitrogen, U.S.), labelled (Ambion, U.S.) and processed by using commercial kits according to the supplier's instructions.
  • the methods of the alkaline heat fragmentation and the following hybridization of the cDNA with the U 133 A and B GeneChip arrays were standard procedure provided by the manufacturer of the microchips (Affymetrix, U.S.).
  • the cell intensity values of the arrays were recorded with a confocal laser scanner (Hewlett Packard, U.S.) and data were analyzed using GeneChip v3.1 software (Affymetrix, U.S.).
  • the expression level for each gene was calculated as normalized average difference of fluorescence intensity as compared to hybridization to mismatched oligonucleotides, expressed as average difference (A.D.). This experiment was performed in triplicate in order to account for biological variation.
  • HMMs Hidden Markov Models
  • the "signal" and “anchor” scores that any input sequence is assigned are fed into a Support Vector Machine (SVM) in a second analysis step (Cristianini N, Shawe-Taylor J. An Introduction to Support Vector Machines and other Kernel-based Learning Methods. Cambridge University Press, Cambridge, England, 2000).
  • SVM Support Vector Machine
  • the SVM was trained on a set of bona fide examples for both classes. On this training set, the SVM obtained the following results on three training sets (signal - anchor - neither).
  • the proteins predicted as extracellular (“signal" or “anchor”) were further evaluated for organ specificity.
  • a search for public domain expressed sequence tags encoding the candidate proteins was carried out and grouped according to tissue source. Only those protein were retained that were expressed in blood vessels and that did not show a strong expression in other secretory organs (e.g. liver, pancreas).
  • Polyclonal antibody to the Insulin Resistance marker Collagen Type IV is generated for further use of the antibody in the measurement of serum and plasma and blood levels of Collagen Type IV by immunodetection assays, e.g. Western Blotting and ELISA.
  • recombinant expression of the protein is performed for obtaining immunogens.
  • the expression is done applying a combination of the RTS 100 expression system and E.coli.
  • the DNA sequence is analyzed and recommendations for high yield cDNA silent mutational variants and respective PCR-primer sequences are obtained using the "ProteoExpert RTS E.coli HY” system. This is a commercial web based service (www.proteoexpert.com).
  • the "RTS 100 E. coli Linear Template Generation Set, His-tag” (Roche Diagnostics GmbH, Mannheim, Germany, Cat.No.
  • His-Collagen Type IV fusion protein Purification of His-Collagen Type IV fusion protein is done following standard procedures on a Ni-chelate column. Briefly, 1 1 of bacteria culture containing the expression vector for the His-Collagen Type IV fusion protein is pelleted by centrifugation. The cell pellet is resuspended in lysis buffer, containing phosphate, pH 8.0, 7 M guanidinium chloride, imidazole and thioglycerole, followed by homogenization using an Ultra-Turrax . Insoluble material is pelleted by high speed centrifugation and the supernatant is applied to a Ni-chelate chromatographic column. The column is washed with several bed volumes of lysis buffer followed by washes with buffer, containing phosphate, pH 8.0 and urea. Finally, bound antigen is eluted using a phosphate buffer containing SDS under acidic conditions.
  • mice 12 week old A/J mice are initially immunized intraperitoneally with 100 ⁇ g Collagen Type IV. This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 ⁇ g Collagen Type IV adsorbed to aluminum hydroxide and 10 9 germs of Bordetella pertussis. Subsequently the last two immunizations are carried out intravenously on the 3rd and 2nd day before fusion using 100 ⁇ g Collagen Type IV in PBS buffer for each.
  • Spleen cells of the mice immunized according to a) are fused with myeloma cells according to Galfre, G., and Milstein, C, Methods in Enzymology 73 (1981) 3-46.
  • ca. l*10 8 spleen cells of the immunized mouse are mixed with 2xlO 7 myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged (10 min at 300 g and 4°C). The cells are then washed once with RPMI 1640 medium without fetal calf serum (FCS) and centrifuged again at 400 g in a 50 ml conical tube.
  • FCS fetal calf serum
  • the sedimented cells are taken up in RPMI 1640 medium containing 10% FCS and sown in hypoxanthine-azaserine selection medium (100 mmol/1 hypoxanthine, 1 ⁇ g/ml azaserine in RPMI 1640 + 10% FCS).
  • Interleukin 6 at 100 U/ml is added to the medium as a growth factor.
  • Collagen Type IV-positive primary cultures are cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter. In this process again interleukin 6 at 100 U/ml is added to the medium as a growth additive.
  • the hybridoma cells obtained are sown at a density of IxIO 5 cells per ml in RPMI
  • 1640 medium containing 10% FCS and proliferated for 7 days in a fermenter (Thermodux Co., Wertheim/Main, Model MCS-104XL, Order No. 144-050).
  • concentrations of 100 ⁇ g monoclonal antibody per ml are obtained in the culture supernatant. Purification of this antibody from the culture supernatant is carried out by conventional methods in protein chemistry (e.g. according to Bruck, C, et al., Methods in Enzymology 121 (1986) 587-695).
  • Collagen Type IV Collagen Type IV
  • complete Freund's adjuvant at the ratio of 1:1 is prepared.
  • Each rabbit is immunized with 1 ml of the emulsion at days 1, 7, 14 and 30, 60 and 90. Blood is drawn and resulting anti-Collagen Type IV serum used for further experiments as described in examples 3 and 4.
  • IgG immunoglobulin G
  • rabbit serum is diluted with 4 volumes of acetate buffer (60 mM, pH 4.0). The pH is adjusted to 4.5 with 2 M Tris-base. Caprylic acid (25 ⁇ l/ml of diluted sample) is added drop-wise under vigorous stirring. After 30 min the sample is centrifuged (13,000 x g, 30 min, 4°C), the pellet discarded and the supernatant collected.
  • the pH of the supernatant is adjusted to 7.5 by the addition of 2 M Tris-base and filtered
  • the immunoglobulin in the supernatant is precipitated under vigorous stirring by the drop-wise addition of a 4 M ammonium sulfate solution to a final concentration of 2 M.
  • the precipitated immunoglobulins are collected by centrifugation (8000 x g, 15 min,
  • the dialysate is centrifuged (13,000 x g, 15 min, 4 0 C) and filtered (0.2 ⁇ m).
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl. Per ml IgG solution 50 ⁇ l Biotin -N-hydroxysuccinimide (3.6 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex 200 (10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl). The fraction containing biotinylated IgG are collected. Monoclonal antibodies have been biotinylated according to the same procedure.
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 ANTaOH, 30 mM NaCl, pH 7.5.
  • Per ml IgG solution 50 ⁇ l digoxigenin-3-O-methylcarbonyl- ⁇ - aminocaproic acid-N-hydroxysuccinimide ester (Roche Diagnostics, Mannheim, Germany, Cat. No. 1 333 054) (3.8 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex® 200 (10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl). The fractions containing digoxigenylated IgG are collected. Monoclonal antibodies are labeled with digoxigenin according to the same procedure.
  • Protein samples enriched and isolated from the medium by Heparin columns were solved in sample buffer consisting of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05 % Tween 20, 1 % SDS, and centrifuged at 12,000 g for 10 min at 4°C.
  • sample buffer consisting of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05 % Tween 20, 1 % SDS, and centrifuged at 12,000 g for 10 min at 4°C.
  • the protein concentration of the supernatant was measured by Bradford using a standard curve constructed from a range of known bovine serum albumin standards.
  • sample buffer 60 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, 25% glycerol, and 14.4 mM 2-mercaptoethanol, pH 6.8
  • sample buffer 60 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, 25% glycerol, and 14.4 mM 2-mercaptoethanol, pH 6.8
  • sample buffer 60 mM Tris-HCl, 2% SDS, 0.1% bromophenol blue, 25% glycerol, and 14.4 mM 2-mercaptoethanol, pH 6.8
  • samples were separated by 12.5% homogenous ExcelGel SDS gels (Amersham Bioscience) and electro transferred onto Nitrocellulose membranes.
  • blocking solution 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20 and 5% non-fat dry milk
  • membranes were incubated with rabbit anti-rat antibody for 2 hrs at room temperature, respectively.
  • membranes were incubated with a horseradish peroxidase conjugated anti-rabbit IgG (H+L), anti-mouse IgGi and anti-mouse IgG2a (Southern Biotechnology Associates, Inc., Birmingham, AL), respectively, for 1 hr at room temperature.
  • H+L horseradish peroxidase conjugated anti-rabbit IgG
  • anti-mouse IgGi and anti-mouse IgG2a Southern Biotechnology Associates, Inc., Birmingham, AL
  • Membranes were washed 3 times for 10 min and antigen-antibody complexes were visualized by an enhanced chemiluminescence's reagent (Western Lightning TM, PerkinElmer Life Sciences, Inc., Boston, MA) on an X-ray film according to the manufacturer's protocol.
  • a sandwich ELISA For detection of Collagen Type IV in human serum or plasma, a sandwich ELISA is developed. For capture and detection of the antigen, aliquots of the anti-Collagen Type IV polyclonal antibody (see Example 2) are conjugated with biotin and digoxigenin, respectively.
  • Streptavidin-coated 96-well microtiter plates are incubated with 100 ⁇ l biotinylated anti-Collagen Type IV polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween 20. After incubation, plates are washed three times with 0.9% NaCl , 0.1% Tween 20. Wells are then incubated for 2 h with either a serial dilution of the recombinant protein (see Example 2) as standard antigen or with diluted plasma samples from patients. After binding of Collagen Type IV, plates are washed three times with 0.9% NaCl , 0.1% Tween 20.
  • Plates are subsequently washed three times with the same buffer.
  • wells are incubated with 100 ⁇ l ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 11685767) and OD is measured after 30-60 min at 405 nm with an ELISA reader.
  • the Biotrin Collagen IV EIA is a quantitative enzyme immunoassay for the assay of Collagen IV in human serum.
  • samples were mixed with labelled anti-collagen IV enzyme conjugate.
  • the mixtures were then transferred to microassay wells coated with anti collagen IV where the collagen IV-enzyme conjugate complexes were captured. After incubating, the wells were washed and an enzyme substate was added.
  • the resultant color intensity was proportional to the amount of collagen IV present in the sample.
  • Assay range was 16-1000 ng/mL (16-1000 microg/L) and total assay time was one hour. The assay was performed according to the manufacturer's instructions.
  • Clinical utility of the novel marker Collagen Type IV was assessed by measuring its levels in 10 diabetic patients depending on injections of exogenous insulin and comparing the levels with those measured in 10 patients with demonstrated normal beta cell function.

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Abstract

L'invention concerne la surveillance de la progression d'une maladie et le diagnostic de la résistance à l'insuline dans des diabètes consistant à mesurer des taux du collagène de type IV dans un échantillon liquide et le criblage de composés destinés à la prévention et/ou au traitement des diabètes.
PCT/EP2006/004066 2005-05-09 2006-05-02 Collagene de type iv utilise comme cible/marqueur pour la resistance a l'insuline Ceased WO2006119886A1 (fr)

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Cited By (3)

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EP3739041A1 (fr) * 2014-03-27 2020-11-18 The Salk Institute for Biological Studies Compositions et procédés pour le traitement du diabète de type 1 et 2 et de troubles apparentés
US11685901B2 (en) 2016-05-25 2023-06-27 Salk Institute For Biological Studies Compositions and methods for organoid generation and disease modeling
US11981931B2 (en) 2015-02-27 2024-05-14 Salk Institute For Biological Studies Reprogramming progenitor compositions and methods of use thereof

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EP1542011A1 (fr) * 2002-09-18 2005-06-15 Kudoh, Norio Procede permettant de mesurer le collagene
EP1544620A1 (fr) * 2003-12-18 2005-06-22 F. Hoffmann-La Roche Ag Quantification par dilution combinatoire en série

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EP1544620A1 (fr) * 2003-12-18 2005-06-22 F. Hoffmann-La Roche Ag Quantification par dilution combinatoire en série

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SCHULMAN I H ET AL: "The C57BL/6J db/db mouse as a model of diabetic glomerulosclerosis in normotensive, obese type 2 diabetic subjects", JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY, vol. 13, no. Program and Abstracts Issue, September 2002 (2002-09-01), & MEETING OF THE AMERICAN SOCIETY OF NEPHROLOGY; PHILADELPHIA, PA, USA; OCTOBER 30-NOVEMBER 04, 2002, pages 536A, XP009071497, ISSN: 1046-6673 *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3739041A1 (fr) * 2014-03-27 2020-11-18 The Salk Institute for Biological Studies Compositions et procédés pour le traitement du diabète de type 1 et 2 et de troubles apparentés
US10912800B2 (en) 2014-03-27 2021-02-09 Salk Institute For Biological Studies Compositions and methods for treating type 1 and type 2 diabetes and related disorders
US12213999B2 (en) 2014-03-27 2025-02-04 Salk Institute For Biological Studies Compositions and methods for treating type 1 and type 2 diabetes and related disorders
US11981931B2 (en) 2015-02-27 2024-05-14 Salk Institute For Biological Studies Reprogramming progenitor compositions and methods of use thereof
US11685901B2 (en) 2016-05-25 2023-06-27 Salk Institute For Biological Studies Compositions and methods for organoid generation and disease modeling
US11760977B2 (en) 2016-05-25 2023-09-19 Salk Institute For Biological Studies Compositions and methods for organoid generation and disease modeling

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