WO1999047565A1 - Anticorps monoclonaux anti-inositolphosphoglyanne - Google Patents

Anticorps monoclonaux anti-inositolphosphoglyanne Download PDF

Info

Publication number
WO1999047565A1
WO1999047565A1 PCT/GB1999/000844 GB9900844W WO9947565A1 WO 1999047565 A1 WO1999047565 A1 WO 1999047565A1 GB 9900844 W GB9900844 W GB 9900844W WO 9947565 A1 WO9947565 A1 WO 9947565A1
Authority
WO
WIPO (PCT)
Prior art keywords
ipg
antibody
ipgs
type
antibodies
Prior art date
Application number
PCT/GB1999/000844
Other languages
English (en)
Inventor
Isabel Varela Nieto
José MATO
Jesus Prieto
Phillip Williams
Thomas William Rademacher
Original Assignee
Rademacher Group Limited
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
Priority claimed from GBGB9805739.1A external-priority patent/GB9805739D0/en
Priority claimed from GBGB9811000.0A external-priority patent/GB9811000D0/en
Application filed by Rademacher Group Limited filed Critical Rademacher Group Limited
Priority to JP2000536756A priority Critical patent/JP2002506631A/ja
Priority to EP99910535A priority patent/EP1062246A1/fr
Priority to AU29467/99A priority patent/AU748080B2/en
Priority to CA002321113A priority patent/CA2321113A1/fr
Publication of WO1999047565A1 publication Critical patent/WO1999047565A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to antibodies capable of specifically binding to inositolphosphoglycans (IPGs) , to methods for making these antibodies and their diagnostic and therapeutic uses, in particular in assays.
  • IPGs inositolphosphoglycans
  • IPG inositol phosphoglycan
  • GPI glycosyl phosphatidylinositol
  • IPGs mediate the action of a large number of growth factors including insulin, nerve growth factor, hepatocyte growth factor, insulin-like growth factor I (IGF-I) , fibroblast growth factor, transforming growth factor ⁇ , the action of IL-2 on B-cells and T-cells, ACTH signalling of adrenocortical cells, igE, FSH and hCG stimulation of granulosa cells, thyrotropin stimulation of thyroid cells, cell proliferation in the early developing ear and rat mammary gland.
  • IGF-I insulin-like growth factor I
  • fibroblast growth factor transforming growth factor ⁇
  • ACTH signalling of adrenocortical cells igE, FSH and hCG stimulation of granulosa cells
  • thyrotropin stimulation of thyroid cells cell proliferation in the early developing ear and rat mammary gland.
  • Soluble IPG fractions have been obtained from a variety of animal tissues including rat tissues (liver, kidney, muscle brain, adipose, heart) and bovine liver. IPG 2 biological activity has also been detected in malaria parasitized red blood cells (RBC) and mycobacteria .
  • RBC malaria parasitized red blood cells
  • the polyclonal antibodies produced using this immunisation protocol react also with the GPI-anchor moieties of GPI-anchor containing proteins.
  • the papers report that this polyclonal antibody inhibits the action of insulin on human placental steroidogenesis, inhibits the action of insulin on muscle cells, inhibits the action of insulin on rat diaphragm and inhibits the action of nerve growth factor on chick cochleovestibular ganglia (CVG) .
  • CVG chick cochleovestibular ganglia
  • the present invention relates to the production of polyclonal and monoclonal antibodies specific for P and 3
  • IPGs inositolphosphoglycans
  • present invention also relates to the uses of anti-IPG antibodies, especially in the diagnosis of diabetes and pre-eclampsia.
  • a central problem concerns the identification of patients at risk of developing type I diabetes. This condition typically arises between the ages of 5 and 11 and is caused by a patient's immune system attacking ⁇ -cells in the pancreas, destroying these cells and causing inflammation. If patients at risk of developing type I diabetes could be identified before this attack takes place, it opens up the possibility of ameliorating or preventing the attack by shutting down the pancreas during the period of risk by providing the patients with insulin or an alternative treatment for diabetes. After the period of risk is over, e.g. after puberty, this treatment could cease, and the patient's pancreas allowed to resume making insulin. Currently, there is no reliable method of identifying such patients.
  • the present invention provides a hybridoma cell line which produces anti- inositolphosphoglycan (IPG) monoclonal antibodies, wherein the cell line is selected from hybridoma cell lines 2F7, 2D1 and 5H6 deposited at European Collection of Cell Cultures (ECACC) under accession numbers 98051201, 98031212 and 98030901.
  • IPG inositolphosphoglycan
  • the present invention provides anti- IPG monoclonal antibodies as obtainable from the above hybridoma cell lines or which are capable of binding to an epitope of an IPG which is bound by a monoclonal antibody produced by one or more of the deposited cell lines.
  • the antibodies were produced following immunisation with A- type IPGs as obtainable from rat liver and yet bind to P-type IPGs as obtainable from human placenta. This surprising result indicates that the antibodies recognise a common epitope on these IPGs.
  • the present invention provides antibodies capable of binding to the same or substantially the same epitope of a P or A- type IPG as one of the above monoclonal antibodies.
  • the antibodies of the invention do not bind to the common reactive determinant (CRD) of GPI anchored proteins. Experiments demonstrating this are set out below in example 6.
  • the antibodies are neutralising antibodies and can be used as antagonists to reduce or inhibit one or more of the biological properties of the P or -type IPGs. 5
  • the present invention provides the above antibodies for use in a method of medical treatment and pharmaceutical compositions comprising the antibodies .
  • the present invention provides the use of the above antibodies for the preparation of a composition for use in a diagnostic assay.
  • the assays are for the diagnosis of pre- eclampsia or diabetes.
  • the use is for the diagnosis of patients at risk of developing type I diabetes.
  • the present invention provides a method for diagnosing patients at risk of developing type I diabetes, the method comprising:
  • A-type IPGs comprising: (a) determining the binding of IPGs in the sample to the anti-IPG antibody.
  • the method comprises the further step of administering insulin and/or P-type IPGs and/or A- type IPGs to a patient shown in the assay to have or be at risk of developing type I diabetes, e.g. for the period of risk.
  • the present invention provides a method for diagnosing pre-eclampsia in a patient, the method comprising: 6
  • the method comprises the further step of administering a P-type IPG antagonist to a patient shown in the assay to have or be at risk of developing pre- eclampsia.
  • the binding of IPGs in the sample to the anti-IPG antibody is compared to the amount of binding that takes place in standards, e.g. from a non- diabetic patient, or from a person known to have a particular type of diabetes, e.g a patient known to have developed type I diabetes or have pre-eclampsia.
  • the assay can be carried out by providing the patient with a carbohydrate load (e.g. glucose) and measuring the IPG response following the load over time.
  • a carbohydrate load e.g. glucose
  • the IPG profiles or levels determined from a patient sample can then be compared with the profiles or levels obtained from standards to make the diagnosis.
  • the antibody is selected from the group of deposited monoclonal anti-IPG antibodies, or antibodies prepared using the methods described herein.
  • the present invention provides the use of an antibody described herein in the immunopurification of P or A- type IPGs. 7
  • the present invention provides a method of producing antibodies specific for P and/or A- type IPGs, the method comprising immunising an animal with one or more soluble P or A-type IPGs to elicit an antibody response, wherein the IPGs are not conjugated to an immunogenic carrier.
  • the inventors have surprisingly found that despite the fact that IPGs are small molecules and carbohydrates, it is possible to raise antibodies by immunising animals with them. Further, the fact that conjugation of the IPGs to a carrier is not required means that epitopes on the IPGs are not blocked or altered by the conjugation reaction, allowing antibodies to be raised to a full complement of epitopes on the IPGs.
  • the method is applicable to the production of both polyclonal and monoclonal antibodies, as is demonstrated by the examples below.
  • the animal is immunised via an intraperitoneal route using a soluble form of the IPG.
  • Figure 1 shows a dose-response curve of absorbance plotted against IPG concentration using anti-IPG antibodies in a Sandwich ELISA test.
  • Figure 2 shows how IPG levels in human serum vary with time after an oral load of glucose (75g) in a control 8 patient without a familiar history of type I diabetes.
  • Figure 3 shows how IPG levels in human serum vary with time after an oral load of glucose (75g) in a patient with a familiar history of type I diabetes
  • Figure 4 shows how IPG levels in human serum vary with time after an oral load of glucose (75g) in a patient with carbohydrate intolerance.
  • Figures 5 and 6 show investigations into the properties of monoclonal antibody 2D1 in chick cochleovestibular ganglia (CVG) culture.
  • Figure 7 shows the results of experiments comparing the binding specificity of monoclonal antibody 5H6 , a polyclonal antibody raised against the IPGs and a polyclonal antibody raised against the cross reactive determinant (CRD) common to GPI anchored proteins.
  • Figure 8 shows the results of a PDH phosphatase assay demonstrating that monoclonal antibody 5H6 is capable of inhibiting the action of P-type IPG from rat liver.
  • Figure 9 shows the results of a pre-eclampsia ELISA assay employing anti-IPG antibodies of the invention to measure urine IPG levels.
  • Figure 10 shows the results of a blinded pre-eclampsia assay using anti-IPG monoclonal antibody 2D1 to measure urine IPG levels.
  • Figure 11 shows the relationship between the IPG assay in pre-eclamptic urine using anti-IPG monoclonal antibody 2D1 and platelet counts .
  • Figure 12 shows the relationship between the IPG assay in pre-eclamptic urine using anti-IPG monoclonal antibody 2D1 and plasma AST activity.
  • A-type mediators modulate the activity of a number of insulin-dependent enzymes such as cAMP dependent protein kinase (inhibits) , adenylate cyclase (inhibits) and CAMP phospho-diesterases (stimulates) .
  • P-type mediators modulate the activity of insulin-dependent enzymes such as pyruvate dehydrogenase phosphatase (stimulates) , glycogen synthase phosphatase (stimulates) and cAMP dependent protein kinase (inhibits) .
  • the A- type mediators mimic the lipogenic activity of insulin on adipocytes, whereas the P-type mediators mimic the glycogenic activity of insulin on muscle. Both A and P-type mediators are mitogenic when added to fibroblasts in serum free media. The ability of the mediators to stimulate fibroblast proliferation is enhanced if the cells are transfected with the EGF-receptor. A- type mediators can stimulate cell proliferation in the CVG.
  • Soluble IPG fractions having A- type and P-type activity have been obtained from a variety of animal tissues including rat tissues (liver, kidney, muscle brain, 10 adipose, heart) and bovine liver.
  • a and P-type IPG biological activity has also been detected in human liver and placenta, malaria parasitized RBC and mycobacteria.
  • the ability of a polyclonal cross -reacting anti- inositolglycan antibody to inhibit insulin action on human placental cytotrophoblasts and BC3H1 myocytes or bovine-derived IPG action on rat diaphragm and chick cochleovestibular ganglia suggests cross -species conservation of many structural features.
  • the prior art includes these reports of A and P-type IPG activity in some biological fractions, the purification or characterisation of the agents responsible for the activity was not disclosed until it was reported in the references below.
  • A- type substances are cyclitol-containing carbohydrates, also containing Zn 2+ ion and optionally phosphate and having the properties of regulating lipogenic activity and inhibiting cAMP dependent protein kinase. They may also inhibit adenylate cyclase, be mitogenic when added to EGF-transfected fibroblasts in serum free medium, and stimulate lipogenesis in adipocytes.
  • P-type substances are cyclitol -containing carbohydrates, also containing Mn + and/or Zn 2+ ions and optionally phosphate and having the properties of regulating glycogen metabolism and activating pyruvate dehydrogenase phosphatase. They may also stimulate the activity of glycogen synthase phosphatase, be mitogenic when added to fibroblasts in serum free medium, and stimulate pyruvate 11 dehydrogenase phosphatase.
  • Methods for obtaining A-type and P-type IPGs are set out in detail in Caro et al, 1997, and in W098/11116 and W098/11117. Methods for obtaining the free GPI precursors of the A and P-type IPGs are set out below.
  • Glycolipids were partially purified from rat liver membranes following the method described in Mato et al (1987) , with minor modifications (Varela-Nieto et al,
  • the membrane fraction was obtained by sequential centrifugation from 300 g of material. Total lipids were obtained by chloroform/methanol extraction followed by the removal of nonpolar lipids. GPI was separated from other phospholipids by sequential acid/base silica gel G60 t.l.c. (see below). Lipids were extracted from the t.l.c. plate with methanol, dried, applied to a Sep-Pak C18 cartridge, eluted with methanol, and dried. GPI was spotted onto the origin of a silica gel G60 t.l.c.
  • GPI molecules were further resolved by double-dimension t.l.c. as described in Clemente et al (1995) and Avila et al (1992) or by using high-performance (HP) -t.l.c. plates developed in chloroform:methanol: ammonium hydroxide:water (40:45:3:5:15, by volume) as described in Gaulton (1991).
  • Lipid migration was calibrated in parallel with phospholipid standards that were detected by staining 12 with iodine.
  • the GPI was incubated with 1 unit of PI-PLC from Bacillus thuringiensis for 3 hours according to the manufacturer's instructions. At the end of the incubation, the reaction was terminated by loading the sample onto a Sep-Pak C18 cartridge. The water eluate (5ml) was dried.
  • Antibodies capable of specifically binding to P and A- type IPGs are disclosed herein. These antibodies can be modified using techniques which are standard in the art. Antibodies similar to those exemplified for the first time here can also be produced using the teaching herein in conjunction with known methods. These methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with the IPG or a fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal.
  • a mammal e.g. mouse, rat, rabbit, horse, goat, sheep or monkey
  • an antibody specific for an IPG may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on 13 their surfaces; for instance see WO92/01047.
  • the library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the IPGs (or fragments) , or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.
  • monoclonal antibody refers to an antibody obtained from a substantially homogenous population of antibodies, i.e. the individual antibodies comprising the population are identical apart from possible naturally occurring mutations that may be present in minor amounts .
  • Monoclonal antibodies can be produced by the method first described by Kohler and Milstein, Nature, 256:495, 1975 or may be made by recombinant methods, see Cabilly et al, US Patent No. 4,816,567, or Mage and Lamoyi in Monoclonal Antibody Production Techniques and Applications, pages 79-97, Marcel Dekker Inc, New York, 1987.
  • a mouse or other appropriate host animal is immunised with the antigen by subcutaneous, intraperitoneal, or intramuscular routes to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the IPG used for immunisation.
  • lymphocytes may be immunised in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell, see Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). Immunisation with 14 soluble IPG and via the intraperitoneal route shown in the examples was surprisingly effective in producing antibodies specific for IPGs.
  • the hybridoma cells thus prepared can be seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium) , which substances prevent the growth of HGPR -deficient cells.
  • Preferred myeloma cells are those that fuse efficiently, support stable high level expression of antibody by the selected antibody producing cells, and are sensitive to a medium such as HAT medium.
  • the monoclonal antibodies of the invention are those that specifically bind to either or both P and A- type IPGs .
  • the epitope bound by the antibodies can be mapped using synthetic compounds known to bind to one of the deposited antibodies, e.g. to see whether an antibody has 15 substantially the same or the same binding specificity as the deposited monoclonal antibodies 2F7, 2D1 or 5H6. This can also be carried out in competitive studies to determine whether a given anti-IPG antibody competes with one of the deposited antibodies for a particular IPG epitope.
  • the present invention includes antibodies which are capable of binding to an IPG epitope bound by an exemplified antibody.
  • this epitope is present on both rat liver A- type IPG and human placenta P-type IPG.
  • the exemplified monoclonal raised using rat liver A- type IPGs are surprisingly effective in assays for the diagnosis of pre-eclampsia, as shown in the examples below.
  • the monoclonal antibody will have an affinity which is greater than micromolar or greater affinity (i.e. an affinity greater than 10 6 mol) as determined, for example, by Scatchard analysis, see Munson & Pollard, Anal. Biochem. , 107:220, 1980.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include Dulbecco's Modified Eagle's Medium or RPM1-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumours in an animal.
  • the monoclonal antibodies secreted by the subclones are 16 suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • Nucleic acid encoding the monoclonal antibodies of the invention is readily isolated and sequenced using procedures well known in the art, e.g. by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies.
  • the hybridoma cells of the invention are a preferred source of nucleic acid encoding the antibodies or fragments thereof. Once isolated, the nucleic acid is ligated into expression or cloning vectors, which are then transfected into host cells, which can be cultured so that the monoclonal antibodies are produced in the recombinant host cell culture.
  • Hybridomas capable of producing antibody with desired binding characteristics are within the scope of the present invention, as are host cells containing nucleic acid encoding antibodies (including antibody fragments) and capable of their expression.
  • the invention also provides methods of production of the antibodies including growing a cell capable of producing the antibody under conditions in which the antibody is produced, and preferably secreted.
  • Antibodies according to the present invention may be modified in a number of ways. Indeed the term “antibody” 17 should be construed as covering any binding substance having a binding domain with the required specificity.
  • the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimics that of an antibody enabling it to bind an antigen or epitope, here a P or A- type inositolphosphoglycan.
  • antibody fragments capable of binding an antigen or other binding partner
  • Fab fragments consisting of the VL, VH, Cl and CHI domains
  • Fd fragment consisting of the VH and CHl domains
  • Fv fragment consisting of the VL and VH domains of a single arm of an antibody
  • dAb fragment which consists of a VH domain
  • isolated CDR regions and F(ab') 2 fragments a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region.
  • Single chain Fv fragments are also included.
  • a hybridoma producing a monoclonal antibody according to the present invention may be subject to genetic mutation or other changes. It will further be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce other antibodies, humanised antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs) , of an antibody to the constant regions, or 18 constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A- 0184187, GB-A- 2188638 or EP-A-0239400. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP- A-0125023.
  • the antibodies of the invention can be formulated in pharmaceutical compositions.
  • An example of this is employing antibodies specific for P-type IPG in the treatment of pre-eclampsia, a condition associated with elevated levels of P-type IPGs. Protocols for the treatment of pre-eclampsia using P-type IPG antagonists are described in copending W098/10791.
  • compositions may comprise, in addition to one or more of the antibodies, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non- toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal 19 or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • the pharmaceutically useful compound according to the present invention is given to an individual in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual.
  • this will be to cause a therapeutically useful effect in the patient, e.g. using the antibodies to antagonise one or more of the biological activities of P and/or A- type IPGs to a beneficial extent.
  • the actual amount of the antibodies administered, and rate and time- course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g.
  • the composition can be administered to provide an initial dose of about 0.01 to 20 mg, more preferably 0.02 to 10 mg, of antibody/kg of patient weight.
  • an initial dose of about 0.01 to 20 mg, more preferably 0.02 to 10 mg, of antibody/kg of patient weight.
  • other dosing regimens and the determination of appropriate amount of the antibodies for inclusion in the compositions can be readily determined by those skilled in the art.
  • Immunoassavs The antibodies described above can be employed in the diagnostic aspects of the invention in a wide variety of different assay formats.
  • the antibodies can be used as binding agents capable of specifically binding to IPGs or as developing agents for determining the fraction of binding sites of a binding agent occupied by analyte after exposure to a test sample.
  • employing the antibodies involves tagging them with a label or reporter molecule which can directly or indirectly generate detectable, and preferably 21 measurable, signals.
  • the linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule. Any method known in the art for separately conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al, Nature 144:945, 1962; David et al, Biochemistry 13:1014, 1974; Pain et al, J. Immunol. Meth. 40:219, 1981; and Nygren, J Histochem. and Cytochem. 30:407, 1982.
  • Suitable fluorochromes include fluorescein, rhodamine, luciferin, phycoerythrin and Texas Red.
  • Suitable chromogenic dyes include diaminobenzidine.
  • Other detectable labels include radioactive isotopic labels, such as 3 H, 14 C, 32 P, 35 S, 126 I, or 99m Tc, and enzyme labels such as alkaline phosphatase, ⁇ -galactosidase or horseradish peroxidase, which catalyze reactions leading to detectable reaction products and can provide amplification of signal.
  • reporter include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise 99/47565
  • These molecules may be enzymes which catalyze reactions that develop or change colour or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors.
  • the antibodies can be employed as binding agents, relying on the fact that the antibodies are capable of specifically binding the IPGs in preference to other substances present in the sample.
  • the binding agent (s) are immobilised on solid support, e.g. at defined locations, to make them easy to manipulate during the assay. This can be achieved using techniques well known in the art such as physisorption or chemisorption, e.g. employing biotin/avidin or biotin/streptavidin to chemically link the antibodies to the solid support.
  • the sample is contacted with the binding agent (s) under appropriate conditions so that P and A- type IPGs present in the sample can bind to the binding agent (s) .
  • the fractional occupancy of the binding sites of the binding agent (s) can then be determined using a developing agent or agents.
  • the developing agents are labelled (e.g. with radioactive, fluorescent or enzyme labels) so that they can be detected using techniques well known in the art.
  • radioactive labels can be detected using a scintillation counter or other radiation counting 99/47565
  • the developing agent (s) can be used in a competitive method in which the developing agent competes with the analyte for occupied binding sites of the binding agent, or non- competitive method, in which the labelled developing agent binds analyte bound by the binding agent or to occupied binding sites. Both methods provide an indication of the fraction of the binding sites occupied by the analyte, and hence the concentration of the analyte in the sample, e.g. by comparison with standards obtained using samples containing known concentrations of the analyte.
  • Diagnostic assays can be carried out with a biological sample from a patient. These samples can be used directly or in some instances may require treatment prior to carrying out the assay, e.g. to remove potentially interfering substances in the sample. Examples of suitable biological samples are blood, urine, sweat, tissue or serum.
  • the present invention concerns a method a diagnosing patients at risk of developing type I diabetes, the method comprises the steps of:
  • step (c) detecting the label of the developing agent specifically binding in step (b) to obtain a value representative of the concentration of the IPGs in the sample.
  • the present invention provides a method for diagnosing pre-eclampsia in a patient, the method comprising:
  • the binding of IPGs in the sample to the anti- IPG antibody is compared to the amount of binding that takes place in controls, e.g. from a non-diabetic, or from a person known to have a particular type of diabetes, e.g a patient known to have developed type I diabetes or pre-eclamptic standards.
  • controls e.g. from a non-diabetic, or from a person known to have a particular type of diabetes, e.g a patient known to have developed type I diabetes or pre-eclamptic standards.
  • this is assessed by initially providing the patient with a glucose load and measuring the IPG response following the glucose load over time.
  • the IPG profiles or levels determined from a sample can then be compared with the profiles or levels obtained from standards to make the diagnosis.
  • the method includes the further step of 25 correlating the value representative of the concentration of the IPGs in the sample to values obtained from known standards to determine whether the patient is at risk from developing type I diabetes or has pre-eclampsia.
  • the antibodies of the present invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays, see Zola, Monoclonal Antibodies: A Manual of Techniques, pp 147-158 (CRC Press, Inc, 1987) .
  • Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the IPG to be detected.
  • the test sample analyte is bound by a first antibody which is immobilised on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three part complex.
  • the second antibody may itself be labelled with a detectable moiety (direct sandwich assays) or may be measured using an anti- immunoglobulin antibody that is labelled with a detectable moiety (indirect sandwich assay) .
  • sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
  • the antibodies of the invention also are useful for in vivo imaging, wherein an antibody labelled with a detectable moiety for example radioisotope and is administered to a host, preferably into the bloodstream, and the presence and location of the labelled antibody in 26 the host is assayed.
  • This antibody may be labelled with any moiety that is detectable in a host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • the antibodies of the invention also are useful as affinity purification agents.
  • the antibodies are immobilised on a suitable support, such as Sephadex ® resin or filter paper, using methods well known in the art.
  • the immobilized antibody then is contacted with a sample containing the IPGs to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the IPGs, which is bound to the immobilised antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 3-5, that will release the IPGs from the antibody.
  • This method can also be used to separate a given IPG family member from a mixture of IPGs by using an antibody capable of specifically binding that IPG in preference to other family members.
  • Inositolphosphoglycan (soluble form) obtained by PI-PLC treatment of GPI purified from rat liver by sequential Thin Layer Chromatography (TLC) was used to immunize New Zealand rabbits and Balb/c mice as described below.
  • human IPGs could be obtained using the 99/47565
  • IPG soluble form
  • CFA complete Freund's adjuvant
  • mice Four female Balb/c 6 weeks old mice were immunised with 60 ⁇ g of IPG (soluble form) in 250 ⁇ l of PBS with 250 ⁇ l of CFA.
  • the antigen-adjuvant emulsion was injected by intraperitoneal (ip) injection.
  • Buffer for ABTS is added to distilled water (1:10 v/v) .
  • lmg of ABTS is added to 1ml of diluted buffer for ABTS.
  • Solvents used are phosphate-buffered saline (PBS) pH 7.2 or PBS-Tween 20 (0.1%).
  • Step 1 IPG (soluble form) is used in an indirect ELISA (initial concentration approximately 60 ⁇ M) .
  • Working dilutions are normally between 1:400 and 1:800.
  • Step 3 Blocking reagent for ELISA is a registered product of Boehringer Mannheim.
  • the content 27g
  • 100 ml redist Water at room temperature while stirring for approx 30 minutes.
  • the concentrate is stable when stored in aliquots at -20°C.
  • dilute one aliquot in a ratio of 1:10 with redistilled water to yield a working solution containing 1% protein mixture (w/v) that was obtained by proteolytic degradation of purified gelatin, in 50mM Tris-HCl, 150mM NaCl, pH ca.7.4. 99/47565
  • Step 5 A purified monoclonal and/or polyclonal antibody are used in an indirect ELISA (initial concentration approx 2mg/ml) .
  • Working dilutions are normally between 1:100 and 1:200, getting final concentrations of 20 ⁇ g/ml and 10 ⁇ g/ml respectively) .
  • incubation time for rapid assays can be two hours at room temperature.
  • Step 7 The anti -rabbit and the anti -mouse antibodies are used at 1:1000 dilution straight from the stock bottle.
  • the stock bottle is stored at 2-8°C. Under these conditions the product is stable for at least six months, after which any remaining solution is discarded.
  • biotinylated reagent is supplied in 0.1 M phosphate-buffered saline of pH7.5 containing 1% (w/v) bovine serum albumin and 0.05% (w/v) sodium azide.
  • Step 9 Streptavidin-biotinylated/HRP is used at 1:500 dilution straight from the stock bottle.
  • the stock bottle is stored at 2-8°C. Under these conditions the product is stable for at least three months, after which any remaining solution is discarded.
  • the complex is supplied in 2ml of phosphate-buffered saline pH7.5 containing 1% (w/v) bovine serum albumin and an antimicrobial agent.
  • Step 11 ABTS buffer is a registered product of
  • the following ELISA protocol is provided as an example of an assay for IPGs employing the deposited monoclonal antibodies.
  • Anti-IPG monoclonal antibody was diluted 1:100 in PBS in added to a F96 Maxisorp Nunc-Immuno plate. The plate was incubated for at least 2 days at 4°C,
  • Blocking reagent for Elisa (Boehringer Mannheim) in distilled water (1.5:10 v/v) was added for 2 hours at room temperature.
  • the plate was washed with PBS-Tween 20 (0,1%) three times .
  • the solvents used were phosphate-buffered saline (PBS) pH7.2 or PBS-Tween 20 (0.1%).
  • step (1) The examples employed purified monoclonal antibody 2D1 in the Sandwich ELISA assays (initial concentration 2.5mg/ml). Working dilutions varied normally between 1:100 and 1:200, with final concentrations of 25 ⁇ g/ml and 12.5 ⁇ g/ml, respectively.
  • the incubation time used in the assay can be varied. For rapid assays, incubation time can vary between two hours at room temperature or overnight at 4°C. However, improved results are obtained when incubation time is at least two days at 4°C. This improvement may be due to the higher incubation times increasing the number of molecules of monoclonal antibody that are bound to the 33 solid phase.
  • Step (3) Blocking reagent for ELISA is a registered product of Boehringer Mannheim.
  • the content 27g was dissolved in 100ml redistilled water at room temperature while stirring for approximately 30mins.
  • the concentrate is stable when stored in aliquots at -20°C.
  • aliquots were diluted in a ratio of 1:10 with redistilled water to yield a working solution containing 1% protein mixture (w/v) that was obtained by proteolytic degradation of purified gelatin, in 50 mM Tris-HCl, 150 mm NaCl, pH ca. 7.4.
  • Step (5) Human serum was probed in several dilutions (1:1, 1:2, 1:4). Working dilutions are normally between 1:2 and 1:4. IPG in several dilutions was used as a standard (positive control) . Wells without serum are used as negative control.
  • the incubation time for rapid assays was two hours at room temperature.
  • Step (7) A purified polyclonal antibody was used in Sandwich ELISA (initial concentration 2 mg/ml) .
  • Working dilutions were between 1:100 and 1:200, with final concentrations of 20 ⁇ g/ml and 10 ⁇ g/ml respectively.
  • Step (9) The anti-rabbit was used at 1:1000 dilution straight from the stock bottle, stored at 28°C. Under these conditions the product is stable for at least six 34 months .
  • biotinylated reagent is supplied in 0.1 M phosphate-buffered saline pH 7.5 containing 1% (w/v) bovine serum albumin and 0.05% (w/v) sodium azide.
  • Step (11) Streptavidin-biotinylated/HRP was used at 1:500 dilution straight from the stock bottle, stored at 2-8°C. Under these conditions the product is stable for at least three months.
  • the complex is supplied in 2ml of phosphate-buffered saline pH 7.5 containing 1% (w/v) bovine serum albumin and an antimicrobial agent.
  • ABTS buffer is a registered product of Boehringer Mannheim.
  • the solution consists of sodium perborate, citric acid and disodium hydrogen phosphate.
  • the solution is stored in aliquots of lml at -20°C. For use, one aliquot is diluted in a ratio of 1:10 with redistilled water.
  • Figure 1 shows a dose-response curve of assay response (absorbance) plotted against IPG concentration, demonstrating that the antibodies can be used to assay for IPGs.
  • FIG. 3 Assay for Type I Diabetes and Controls
  • the protocol set out in example 3 was used to measure IPG levels in a different patients in response to a glucose 35 load (75g) .
  • Figures 2 to 4 plot the changes in IPG levels in the patients over time after the glucose load.
  • the profiles shown in the figures and/or the levels of the IPGs can be used in the diagnosis of the risk of developing type I diabetes by comparing them with the profile or level obtained from a sample from a patient suspected of being at risk of developing type I diabetes.
  • examining the change in IPG levels has the advantage that it isolates the change in IPG level caused by the glucose load from the effect of IPGs acting as second messengers for other growth factors in the patient.
  • Figure 2 shows the response obtained in a control patient with no familial history of diabetes.
  • the IPG level in the patient rose to a maximum around 2 hours after the load, and then fell again to the background level.
  • Figure 4 shows a second control profile obtained from a patient with carbohydrate intolerance, i.e. who was unable to metabolise the glucose via insulin and subsequent IPG production.
  • the IPG profile following the glucose load is approximately flat.
  • Figure 3 shows a profile obtained from a patient with a familial history of type I diabetes. No elevation in IPG is observed indicating that there has been an inadequate release of insulin in response to the glucose challenge.
  • CVGs were isolated from E3.5 chicken embryos as described in Varela-Nieto et al, 1991, and cultured for 24 hours in 4 -well multidishes (NUNC) in 0.25 ml of M199 culture medium with Earle's salts, 2mM L-glutamine, 25mM HEPES and antibiotics. Incubations were carried out at 37°C in a humidified atmosphere with 5% C0 2 .
  • the photomicrographs of figure 5 show CVG proliferation under the following conditions: control medium with no additions (A) , 25 ⁇ g/ml 2D1 antibody (B) , InM IGF-I alone (C) , 1 nM IGF-1 plus 25 ⁇ g/ml 2D1 (D) .
  • control medium with no additions A
  • B 25 ⁇ g/ml 2D1 antibody
  • C InM IGF-I alone
  • D 1 nM IGF-1 plus 25 ⁇ g/ml 2D1
  • Figure 6A shows percent of inhibition of 2D1 antibody on CVG cultured in presence of IGF- I.
  • CVGs were isolated as described above. The DNA synthesis was determined by culturing the CVG explants in M199 medium containing 10 ⁇ Ci/ml of [H] thymidine of 24 hours. 37
  • Radioactivity incorporated by the explants was extracted with 10% trichloroacetic acid and quantified by scintillation counting.
  • A Percent of inhibition in CVGs cultured with 1 nM IGF-I in presence of increasing concentrations of 2D1 antibody from 25 to 100 ⁇ g/ml.
  • B Acid-precipitable [ 3 H] thymidine uptake into CVGs cultured in parallel experiments with the additions indicated in Figure 5 (A-H) .
  • the exogenous addition of 100 ⁇ M of synthetic analogue C3 was unable to rescue the 2D1 inhibitory effect on CVG growth induced by IGF-I (I) .
  • Data are representative of at least 3 different experiment with an average of 4 CVGs per condition.
  • figure 7 shows that antibody 5H6 does not cross react with either soluble or membrane bound VSG, whereas both polyclonal antibodies react strongly. This property distinguishes the antibodies of the invention from cross reacting polyclonal antibodies. This in turn suggests that polyclonal antibodies will not specifically detect IPGs, but are a valuable reagent in ELISA sandwich assays which require two anti-IPG antibodies, one of which is very specific (monoclonal) and the other of general reactivity (polyclonal) .
  • FIG. 8 shows that monoclonal antibody 5H6 is able to inhibit the action of P-type IPG from rat liver in a P- type phosphatase assay.
  • this and similar antibodies could be used as P-type IPG antagonists, e.g. in the treatment of conditions associated with elevated levels of P-type IPGs such as pre-eclampsia.
  • Example 8 Assay for Urine IPG Levels (e.g. for use in the diagnosis of pre-eclampsia)
  • the following assay was carried out using anti-IPG monoclonal 2Dl-the capture antibody and polyclonal rabbit IgG anti-IPG as the secondary antibody.
  • the assay was carried out using samples from pre-eclamptic and control pregnant patient urine samples.
  • a Maxisorb plate was coated with 50ml of 2D1 antibody at 2.5mg/ml in PBS and incubated overnight at 4°C. The plate was then blocked with 200ml of blocking reagent and incubated for lh at 37°C in a sealed container floating 39 on a waterbath.
  • Figure 9 shows results from the assay comparing two samples from pre-eclamptic patients with non-pre- eclamptic controls.
  • Figure 10 shows the results obtained from a blinded study of 24 samples, 12 from pre-eclamptic patients. This shows that the assay successfully identified 10/12 of the pre-eclamptic samples with no false positives.
  • Figure 11 shows the correlation between platelet counts and the reaction of 2D1 monclonal antibody with urine from pre-eclamptic women.
  • the inverse correlation is significant p ⁇ 0.05.
  • pre-eclamptic women there is a 99/47565
  • Figure 12 shows the correlation between plasma aspartic transaminase (AST) and the binding reactivity of 2D1 to urine from pre-eclamptic women.
  • AST plasma aspartic transaminase
  • W098/011116 and W098/011117 (Rademacher Group Limited)

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Diabetes (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Endocrinology (AREA)
  • Emergency Medicine (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des anticorps anti-IPG, et plus particulièrement des anticorps monoclonaux produits par les lignées cellulaires hybridomes 2F7, 2D1, et 5H6. L'invention concerne également l'utilisation des anticorps susmentionnés et d'autres anticorps similaires, afin de diagnostiquer et de traiter la prééclampsie ou les diabètes, en particulier les diabètes du type I. L'invention concerne enfin une méthode de production desdits anticorps anti-IPG, par immunisation d'un animal avec un IPG non conjugué à un excipient immunogène.
PCT/GB1999/000844 1998-03-18 1999-03-18 Anticorps monoclonaux anti-inositolphosphoglyanne WO1999047565A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2000536756A JP2002506631A (ja) 1998-03-18 1999-03-18 抗イノシトールホスホグリカンモノクローナル抗体
EP99910535A EP1062246A1 (fr) 1998-03-18 1999-03-18 Anticorps monoclonaux anti-inositolphosphoglyanne
AU29467/99A AU748080B2 (en) 1998-03-18 1999-03-18 Anti-inositolphosphoglycan monoclonal antibodies
CA002321113A CA2321113A1 (fr) 1998-03-18 1999-03-18 Anticorps monoclonaux anti-inositolphosphoglyanne

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GBGB9805739.1A GB9805739D0 (fr) 1998-03-18 1998-03-18
GB9805739.1 1998-03-18
GBGB9811000.0A GB9811000D0 (fr) 1998-05-21 1998-05-21
GB9811000.0 1998-05-21

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09646468 A-371-Of-International 2000-10-26
US43985103A Continuation 1998-03-18 2003-05-15

Publications (1)

Publication Number Publication Date
WO1999047565A1 true WO1999047565A1 (fr) 1999-09-23

Family

ID=26313300

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1999/000844 WO1999047565A1 (fr) 1998-03-18 1999-03-18 Anticorps monoclonaux anti-inositolphosphoglyanne

Country Status (5)

Country Link
EP (1) EP1062246A1 (fr)
JP (1) JP2002506631A (fr)
AU (1) AU748080B2 (fr)
CA (1) CA2321113A1 (fr)
WO (1) WO1999047565A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000039285A2 (fr) * 1998-12-24 2000-07-06 University College London Proteines d phospholipase specifiques du glycosyl phosphatidy linositol et leurs utilisations
WO2001086292A2 (fr) * 2000-05-12 2001-11-15 Rodaris Pharmaceutical Limited Dosages biologiques

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996029425A1 (fr) * 1995-03-21 1996-09-26 University College London Imitateurs d'hormones et de facteurs de croissance
WO1998011117A1 (fr) * 1996-09-11 1998-03-19 Rademacher Group Limited Hydrates de carbone contenant du cyclitol obtenus a partir de tissus humains qui regissent le metabolisme du glycogene
WO1998011116A1 (fr) * 1996-09-11 1998-03-19 Rademacher Group Limited Hydrates de carbone contenant cyclitol, qui proviennent de tissus humains et regulent l'activite lipogene
WO1998010791A1 (fr) * 1996-09-11 1998-03-19 Rademacher Group Limited Substances et methodes destinees au diagnostic et au traitement de la preeclampsie et du diabete
WO1998011435A1 (fr) * 1996-09-11 1998-03-19 Rademacher Group Limited Substances et methodes relatives au diagnostic et au traitement du diabete et de l'obesite

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996029425A1 (fr) * 1995-03-21 1996-09-26 University College London Imitateurs d'hormones et de facteurs de croissance
WO1998011117A1 (fr) * 1996-09-11 1998-03-19 Rademacher Group Limited Hydrates de carbone contenant du cyclitol obtenus a partir de tissus humains qui regissent le metabolisme du glycogene
WO1998011116A1 (fr) * 1996-09-11 1998-03-19 Rademacher Group Limited Hydrates de carbone contenant cyclitol, qui proviennent de tissus humains et regulent l'activite lipogene
WO1998010791A1 (fr) * 1996-09-11 1998-03-19 Rademacher Group Limited Substances et methodes destinees au diagnostic et au traitement de la preeclampsie et du diabete
WO1998011435A1 (fr) * 1996-09-11 1998-03-19 Rademacher Group Limited Substances et methodes relatives au diagnostic et au traitement du diabete et de l'obesite

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CARO H N ET AL: "ISOLATION AND PARTIAL CHARACTERISATION OF INSULIN-MIMETIC INOSITOL PHOSPHOGLYCANS FROM HUMAN LIVER", BIOCHEMICAL AND MOLECULAR MEDICINE, vol. 61, 1 August 1997 (1997-08-01), pages 214 - 228, XP002050247 *
REPRESA, J ET AL: "Glycosyl-phosphatidylinositol/inositol phsphoglycan: A signaling system for the low-affinity nerve growth factor receptor", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 88, 1991, pages 8016 - 8019, XP002107916 *
ROMERO G. ET AL: "Anti-inositolglycan antibodies selectively block some of the actions of insulin in intact BC3H1 cells", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 87, 1990, pages 1476 - 1480, XP002107915 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000039285A2 (fr) * 1998-12-24 2000-07-06 University College London Proteines d phospholipase specifiques du glycosyl phosphatidy linositol et leurs utilisations
WO2000039285A3 (fr) * 1998-12-24 2000-11-16 Univ London Proteines d phospholipase specifiques du glycosyl phosphatidy linositol et leurs utilisations
WO2001086292A2 (fr) * 2000-05-12 2001-11-15 Rodaris Pharmaceutical Limited Dosages biologiques
WO2001086292A3 (fr) * 2000-05-12 2002-06-20 Rodaris Pharmaceutical Ltd Dosages biologiques

Also Published As

Publication number Publication date
CA2321113A1 (fr) 1999-09-23
JP2002506631A (ja) 2002-03-05
AU2946799A (en) 1999-10-11
EP1062246A1 (fr) 2000-12-27
AU748080B2 (en) 2002-05-30

Similar Documents

Publication Publication Date Title
DE69936780T2 (de) Antikörper gegen eine lar-phosphatase untereinheit
DE60025008T2 (de) Bestimmung von adrenomedullin-bindenden proteinen
US9783600B2 (en) Apolipoprotein C3 (ApoCIII) antagonists and methods of their use to remove ApoCIII inhibition of lipoprotein lipase (LPL)
Merlie et al. Acetylcholine receptor degradation in adult rat diaphragms in organ culture and the effect of anti-acetylcholine receptor antibodies.
CN106046158A (zh) 针对人前列腺素e2 受体ep4 的抗体
JP2007536212A (ja) レプチンアンタゴニストおよびレプチン定量測定方法
KR20080106157A (ko) Ｆａｓ 결합 항체
JPH09507578A (ja) インスリン受容体チロシンキナーゼインヒビターに対するアンタゴニスト
JPWO2008018472A1 (ja) 新規モノクローナル抗体およびその用途
EP0443599B1 (fr) Méthode pour la détection des des antigènes associés aux tumeurs
US7732147B2 (en) Neurotransmission disorders
JPH09507754A (ja) 哺乳類悪性細胞の選択的メチオニン飢餓方法
JP2001519894A (ja) リソソーム蓄積症の早期検出法
US8038997B2 (en) Anti-marinobufagenin antibodies and methods for their use
AU748080B2 (en) Anti-inositolphosphoglycan monoclonal antibodies
Yamamoto et al. Immunohistochemistry for anti-diabetes drug, alogliptin using a newly prepared monoclonal antibody: Its precise localization in rat small intestine
US20040192586A1 (en) Materials and methods relating to anti-inositolphosphoglycan antibodies
US5939269A (en) Antagonists to insulin receptor tyrosine kinase inhibitor
US6777394B1 (en) Methods and compositions for regulating imidazoline receptors
JP5749643B2 (ja) 診断及び予後の方法を実施するためのｉｒａｐタンパク質の使用
JP2792651B2 (ja) ヒト膵臓癌に対するモノクローナル抗体
JPH10508831A (ja) ビタミンｂ１２／トランスコバラミンｉｉレセプターおよび結合部位に対する抗レセプターおよび増殖阻止剤
DE60216482T2 (de) Zusammensetzungen und verfahren zum bestimmen von aa4rp
Fujimiya et al. Immuno-electron microscopic study of differential localization of motilin and serotonin in the rabbit duodenal epithelium
CA2321734A1 (fr) Substances et methodes pour identifier des composes mimetiques d'inositolphosphoglycanne

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK 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 MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

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

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1999910535

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2321113

Country of ref document: CA

Ref country code: CA

Ref document number: 2321113

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 29467/99

Country of ref document: AU

NENP Non-entry into the national phase

Ref country code: KR

WWE Wipo information: entry into national phase

Ref document number: 09646468

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1999910535

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWG Wipo information: grant in national office

Ref document number: 29467/99

Country of ref document: AU

WWW Wipo information: withdrawn in national office

Ref document number: 1999910535

Country of ref document: EP