WO2006063736A1 - L'antigene cd31 marqueur de cibles pour detecter la resistance a l'insuline - Google Patents

L'antigene cd31 marqueur de cibles pour detecter la resistance a l'insuline Download PDF

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Publication number
WO2006063736A1
WO2006063736A1 PCT/EP2005/013199 EP2005013199W WO2006063736A1 WO 2006063736 A1 WO2006063736 A1 WO 2006063736A1 EP 2005013199 W EP2005013199 W EP 2005013199W WO 2006063736 A1 WO2006063736 A1 WO 2006063736A1
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Prior art keywords
pecam
insulin resistance
compound
protein
diabetes
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PCT/EP2005/013199
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English (en)
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Peter Berndt
Stefan Evers
Stefan Foser
Michael Fountoulakis
Mitchell Lee Martin
Elena Sebokova
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F. Hoffmann-La Roche Ag
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Publication of WO2006063736A1 publication Critical patent/WO2006063736A1/fr

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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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • 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

  • CD31 antigen 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 normoglycaemic 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.
  • the cell adhesion molecule PECAM-I (CD31) is a 130 kDa member of the immunoglobulin (Ig) gene superfamily that is constitutively expressed at high concentration at endothelial cell intercellular junctions and at moderate density on the surface of circulating leukocytes and platelets. No evidence existed so far for a correlation between levels of secreted PECAM-I in serum and diabetes.
  • 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 of secreted PECAM-I.
  • novel target and/or marker PECAM-I maybe used for diagnostic, monitoring as well as for screening purposes.
  • PECAM-I is a type I transmembrane protein fragments of the sequence of Seq ID No. 1 have to be generated in order for a secreted form to appear in plasma,.
  • the target used for the methods of the present invention, or the markers detectable by the methods of the present invention also includes soluble fragments of Seq ID No. 1.
  • Such soluble fragments of PECAM-I are well known (e.g. Goldberger et al., J. Biol. Chem. 1994, 269, 25, 17183-17191; Serebruany et al., Cardiology 1999, 91, 50-55; Soeki et al., Int. J. Cardiol. 2003, 90, 261-268).
  • PECAM-I and “protein PECAM-I”, as used herein, are understood to include soluble fragments of Seq ID No. 1 as well as the protein of Seq ID No. 1 or mutants thereof which are at least 90 % homologous to Seq ID No. 1.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one polypeptide for optimal alignment with the other polypeptide or nucleic acid molecule).
  • the amino acid residues or nucleotides at corresponding amino acid positions are then compared. When a position in one sequence is occupied by the same amino acid residue as the corresponding position in the other sequence, then the molecules are homologous at that position.
  • amino acid “homology” is equivalent to amino acid “identity”.
  • percent homology between the two sequences is a function of the number of identical positions shared by the sequences (Le., percent homology equals the number of identical positions/total number of positions times 100).
  • the marker PECAM-I consists of any fragment or mutant or native form of Seq ID No. 1 which can be detected by the ELISA described in Example 4 or 5 or by any one of the assays described in Goldberger et al., J. Biol. Chem. 1994, 269, 25, 17183-17191; Serebruany et al., Cardiology 1999, 91, 50-55; Soeki et al., Int. J. Cardiol. 2003, 90, 261-268.
  • 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 PECAM-I 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 PECAM-I and correlating the level of PECAM-I 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 PECAM-I under conditions appropriate for formation of a complex between said binding agent and PECAM-I, 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 PECAM-I under conditions appropriate for formation of a complex between said binding agent and PECAM-I, 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 a method of screening for a compound which interacts with PECAM-I, comprising the steps of a) contacting protein PECAM-I with a compound or a plurality of compounds under compositions which allow interaction of said compound or a plurality of compounds with PECAM-I; and b) detecting the interaction between said compound or plurality of compounds with said polypeptide.
  • the present invention provides a 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 PECAM-I; and b) measuring the activity of protein PECAM-I; wherein a compound which inhibits or stimulates the activity of protein PECAM-I is a compound that may prevent and/or inhibit and/or attenuate Insulin Resistance.
  • said method additionally comprises the step of immobilizing protein PECAM-I prior to step a) or between steps a) and b).
  • the term "activity" as used herein relates to the ability of PECAM-I to interact with itself, or with ligand such as integrin avb3, CD38, 120 kDa ligand on T cells and heparin-dependent proteoglycans.
  • Assays to measure activity of PECAM-I are well known in the art (eg. As described in Sun et al, J. Biol. Chem. 1996, 271 (19), p. 11090- 11098; Piali et al., J. Cell Biol. 1995, 130(2), p. 451-460; Deaglio et al., J. Immunol. 1998, 160, p. 395-402; Prager et al., J. Exp. Med. 1996, 184, p. 41-50; DeLisser et al., J. Biol. Chem. 1993, 268 (21), p. 16037-16046).
  • the present invention also includes cell-free assays.
  • assays involve contacting a form of PECAM-I (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.
  • PECAM-I e.g., full-length polypeptide, a biologically active fragment of said polypeptide, or a fusion protein comprising all or a portion of said polypeptide
  • 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-IOO, 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-oct
  • 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 micro- centrifuge 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.
  • glutathione sepharose beads Sigma Chemical; St. Louis, Mo.
  • 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).
  • 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.
  • Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention.
  • either 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).
  • biotinylation kit Pierce Chemicals; Rockford, 111.
  • antibodies reactive with a polypeptide or binding molecules, but which do not interfere with binding of the polypeptide of the invention to its binding molecule 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 PECAM-I 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 PECAM-I secreted from a host is changed.
  • a host maybe 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 PECAM-I 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 to methods known from the art PECAM-I is specifically measured from this liquid sample by use of a specific binding agent.
  • a specific binding agent is, e.g., a receptor for PECAM- 1 or an antibody to PECAM-I.
  • specific is used to indicate that other biomolecules present in the sample do not significantly bind to the binding agent specific for PECAM-I. A level of less than 5% cross-reactivity is considered not significant.
  • a specific binding agent preferably is an antibody reactive with PECAM-I.
  • 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 PECAM-I in a method according to the present invention is yet another preferred embodiment.
  • PECAM-I has been identified as a marker which is useful in the diagnosis of Insulin Resistance
  • alternative ways maybe 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 PECAM-I 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 PECAM-I under conditions appropriate for formation of a binding agent PECAM- 1-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 PECAM-I -complex all described in detail in relevant textbooks (cf., e.g., Tijssen P., supra, or Diamandis, et al., eds. (1996) Immunoassay, Academic Press, Boston).
  • PECAM-I is detected in a sandwich type assay format.
  • a first specific binding agent is used to capture PECAM-I 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.
  • PECAM-I can be measured from a liquid sample obtained from an individual sample. No tissue and no biopsy sample is required to apply the marker PECAM- 1 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.
  • Antibodies to PECAM-I 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 PECAM-I is used in a qualitative (PECAM-I present or absent) or quantitative (PECAM-I amount is determined) immunoassay.
  • the present invention relates to use of protein PECAM-I 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 PECAM- 1 as measured from a bodily fluid of an individual marks the presence of Insulin Resistance.
  • the use of protein PECAM-I itself represents a significant progress to the challenging field of Insulin Resistance diagnosis. Combining measurements of PECAM-I 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 PECAM-I 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.
  • Diagnostic reagents in the field of specific binding assays usually are best provided in the form of a kit, 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 PECAM-I and auxiliary reagents for measurement of PECAM-I.
  • One way of assessing clinical utility of the novel marker PECAM- 1 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.
  • ROC receiver-operating characteristics
  • 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)] .
  • 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.
  • VECs Human Vascular Endothelial Cells
  • the HUVECs was cultured in pi medium for 48h. After 48h the cells were harvested by scraping and the total cellular RNA was extracted with RNA-Bee . From each sample 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 PECAM-I is generated for further use of the antibody in the measurement of serum and plasma and blood levels of PECAM-I 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-PECAM-1 fusion protein Purification of His-PECAM-1 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-PECAM-1 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 PECAM-I. This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 ⁇ g PECAM-I adsorbed to aluminum hydroxide and 10 9 germs ofBordete ⁇ la pertussis. Subsequently the last two immunizations are carried out intravenously on the 3rd and 2nd day before fusion using 100 ⁇ g PECAM-I in PBS buffer for each. b) Fusion and cloning
  • 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. I 1+ IO 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.
  • After ca. 10 days the primary cultures are tested for specific antibody.
  • PECAM-I -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 0 et al., Methods in Enzymology 121 (1986) 587-695).
  • a fresh emulsion of the protein solution (100 ⁇ g/ml protein PECAM-I) and 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-PECAM-1 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 (0.2 ⁇ m).
  • 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, 4°C).
  • the supernatant is discarded.
  • the pellet is dissolved in 10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl and exhaustively dialyzed.
  • the dialysate is centrifuged (13,000 x g, 15 min, 4°C) and filtered (0.2 ⁇ m).
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 ANaOH, 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 ANaOH, pH 7.5, 30 mM NaCl). The fraction containing biotinylated IgG are collected. Monoclonal antibodies have been biotinylated according to the same procedure. Digoxygenylation of polyclonal rabbit IgG
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, 30 mM NaCl, pH 7.5.
  • Per ml IgG solution 50 ⁇ l digoxigenin-3-O-methylcarbonyl- ⁇ - aminocaproic add-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 ZNaOH, 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 are 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 is 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
  • samples are 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 mine
  • membranes are incubated with rabbit anti-rat antibody for 2 hrs at room temperature, respectively.
  • membranes are 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 are washed 3 times for 10 min and antigen-antibody complexes are 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 PECAM- 1 in human serum or plasma, a sandwich ELISA is developed. For capture and detection of the antigen, aliquots of the anti-PECAM-1 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-PECAM-1 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 PECAM-I, plates are washed three times with 0.9% NaCl , 0.1% Tween 20.
  • wells are incubated with 100 ⁇ l of digoxigenylated anti-PECAM-1 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. Thereafter, plates are washed three times to remove unbound antibody. In a next step, wells are incubated with 20 mU/ml anti-digoxigenin-POD conjugates
  • PECAM-I was detected in plasma of insulin-resistant and insulin-sensitive individuals using the CD31/PECAM-1 kit from DIACLONE.
  • the ELISA was perfomed according to the manufacturer's instructions.
  • the assay protocol was as follows: Standard and controls were prepared as instructed by the manufacturer. 100 ⁇ l of sample were added to sample wells. Biotinylated anti-CD31 was diluted according to the manufacturer's instructions. 50 ⁇ l of diluted biotinylated anti-CD31 was added to all wells. The plate was covered and incubated for 2 hours at room temperature.
  • the cover was removed and the plate washed as follows: 1) The liquid was aspirated from each well; 2) 0.3 ml of washing solution (provided with the kit) was dispensed into each well; 3) the content of each well was aspirated again; 4) steps 2) and 3) were repeated two times.
  • the streptavidin-HRP solution was prepared just before use. Then, 100 ⁇ l of streptavidin- HRP solution was distributed to all wells. The plate was covered and incubated for 30 mins at room temperature. The cover was removed, the wells emptied and the n washed as described previously. Then, 100 ⁇ l of ready-to-use TMB substrate solution was pipetted into all wells and incubated in the dark for 12-15 minutes at room temperature.
  • the mean concentration of PECAM-I measured in plasma of 17 insulin resistant individuals was 4.3824 ng/ml +/- 2.0686 ng/ml, and in 17 insulin sensitive individuals 2.8706ng/ml +/- 1.3032 ng/ml.

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Abstract

L'invention porte: sur le suivi de la progression de la maladie et le diagnostic de la résistance à l'insuline chez les diabétiques, par mesure des niveaux de PECAM-1 dans un échantillon liquide, et sur le criblage de nouveaux composés utiles pour la prévention et/ou le traitement du diabète.
PCT/EP2005/013199 2004-12-14 2005-12-09 L'antigene cd31 marqueur de cibles pour detecter la resistance a l'insuline WO2006063736A1 (fr)

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Cited By (3)

* 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
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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Cited By (5)

* 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
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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