WO1997010504A1 - Dosages radioimmunologiques par anticorps des propeptide/prohormone convertases pc1/3 et pc2 - Google Patents

Dosages radioimmunologiques par anticorps des propeptide/prohormone convertases pc1/3 et pc2 Download PDF

Info

Publication number
WO1997010504A1
WO1997010504A1 PCT/US1996/014800 US9614800W WO9710504A1 WO 1997010504 A1 WO1997010504 A1 WO 1997010504A1 US 9614800 W US9614800 W US 9614800W WO 9710504 A1 WO9710504 A1 WO 9710504A1
Authority
WO
WIPO (PCT)
Prior art keywords
pci
antibody
amount
standardized
reactivity
Prior art date
Application number
PCT/US1996/014800
Other languages
English (en)
Inventor
Bryan D. Noe
John K. Mcdonald
Original Assignee
Emory University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Emory University filed Critical Emory University
Priority to AU70724/96A priority Critical patent/AU7072496A/en
Publication of WO1997010504A1 publication Critical patent/WO1997010504A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6454—Dibasic site splicing serine proteases, e.g. kexin (3.4.21.61); furin (3.4.21.75) and other proprotein convertases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • PCs Prohormone/Propeptide Convertases
  • PCl/3 and PC2 are expressed predominantly within endocrine and neuroendocrine cells and tissues, while furin, PACE4 and PC6 isoforms are expressed ubiquitously.
  • PC6A has been localized only within a subset of endocrine and non- endocrine cells (e.g. pancreatic islets and gut endocrine cells) , while PC4 is expressed primarily within testicular germ cells. See Bresnahan P.A. et al (1990) J " . Cell Biol . Ill :2851-2859 ; Korner, J. et al. (1991) Proc . Nat . Acad. Sci . USA ££:6834-6838; Mackir, R.B. et al. (1987) J. Biol . Chem . 262 :6453-6456 ? Nakayama K. et al. (1991) J. Biochem . (Tokyo) 2JL2. : 803-806.
  • the physiological importance of the mammalian PCs is exemplified by the broad spectrum of precursors they process.
  • the list includes peptide hormones, growth factors, hormone receptors, and envelope giycoproteins of viral pathogens.
  • Hypersecretion of the PCs can also serve as the basis for the application of the RIAs we have developed to detect peptide hormone secreting tumors .
  • Two examples of neuroendocrine tumors that could release excessive amounts of PCs are gastrinomas and insulinomas, both of which hypersecrete peptide hormones, gastrin and insulin respectively, that are derived from larger inactive precursors. Both hormones have broad physiologic effects and are implicated in the generation of secondary complications associated with the presence of these tumors.
  • Another syndrome in which human PC-specific RIAs can have great usefulness clinically is to aid in the diagnosis of familial hyperproinsulinemias or related endocrinopathies.
  • proinsulin In hyperproinsulinemic individuals, proinsulin is not processed properly at both of its endoproteolytic cleavage sites. The resulting product which is released from the beta cells is partially cleaved proinsulin. Because the insulin receptors on insulin sensitive target cells do not respond to these modified forms of proinsulin, afflicted patients exhibit symptoms of insulin deficiency and often develop the characteristics of Type II diabetes mellitus. Since hyperglycemia is a component of the physiologic consequences in these individuals, their beta cells respond by synthesizing and releasing extremely high levels of the modified proinsulin.
  • the etiology of this clinical syndrome can have its basis in synthesis of proinsulin with one or more amino acid substitutions in the basic pair cleavage site, thus preventing PC-mediated cleavage at this site.
  • production of insufficient amounts of the appropriate PCs, or production of mutated PCs that are enzymatically inactive can have effects that result in the same clinical picture in patients. Because essentially all peptide hormones and neuropeptides are synthesized by precursors, a reduction in levels of expression, or defects in the PCs that render them enzymatically inactive can result in hypersecretion of partially processed prohor ones which are metabolically ineffective.
  • the PC RIAs described herein provide invaluable tools to determine whether the cause of reduced cleavage resulted from decreased expression of the cleavage enzymes. From the discussion above, it should be clear that the RIAs that we have developed are useful in clinical diagnoses of existing conditions and also provide a valuable tool to screen patients at risk to develop hormonal imbalance.
  • One of the approaches employed in such studies is to introduce antisense DNA into cells that co-express the PCs and their substrate (s) in an attempt to neutralize PC-specific mRNA through hybridization with the antisense nucleotide that is expressed.
  • An expected consequence is reduction of translation of the mRNA that combines with the antisense molecule, thereby reducing the amount of PC expression and activity.
  • an essential control in the performance of such experiments is to monitor the actual levels of PC that are expressed in cells transfected with expression vector that either contains (experimental) or lacks (control) the antisense coding region. Such data provide an assessment of the relative amount that the expression of the targeted PC is reduced in the antisense treated cells.
  • the invention provides highly specific antibodies for human PCI/3 and for PC2.
  • PCI/3 is used throughout since it has been termed PCI, PC3, or PCI/3 in the literature.
  • RIA radioimmune assay
  • the cross-reactivity of the anti-PCl/3 antibody was less than .002% for hPC2- ⁇ 25 the region of PC2 corresponding to amino acids 112-136, less than .0006% for hPC2-C25, the region of PC2 corresponding to amino acids 136-159, and less than .001% for a fusion protein of glutathione-S-transferase (GST) and PC2.
  • the cross- reactivity of the anti-PC2 antibody was less than 0.8% for hPCl/3-N23, a peptide containing amino acids 114-136 of human PCI/3, less than 0.03% for hPCl/3-C22, amino acids 137-158 of PCI/3, and less than 0.2% for a fusion protein of GST and PCI/3.
  • the comparison in each case was the reactivity with hPCl/3-N23 in the case of anti PCI/3 or hPC2-N25 in the case of anti-PC2.
  • Normal ranges for PCI/3 and PC2 in human serum have now been determined, using the RIA. These are 220.7 ⁇ 82.9pg/ml for PCI/3 and 103.7 ⁇ 27.7pg/ml for PC2.
  • Clinical diseases associated with defective hormone processing have been identified, including a polyendocrine disease [O'Rahilly, S. et al. (1995) Abstr. 77th Ann. Mtg. Endocrine Soc. p. 597, Abstract p3-513] and non-insulin dependent diabetes mellitus (NIDDM) [Yoshida, H. et al . (1995) Diabetes -1:389-393] .
  • Clinical assay immunoassay kits for measuring PCI/3 and for PC2 levels in serum are therefore provided as useful components for diagnosis of disease conditions associated with defective hormone processing.
  • Such defective processing can be manifested either by abnormally high levels of one or both PCI/3 and PC2, or by abnormally low levels of one or both enzymes.
  • the invention provides a diagnostic method for detecting a clinical disease associated with defective hormone processing, including, but not limited to, neuroendocrine tumor, hyperproinsulinemia, hyperproglucagonemia, polyendocrine disease, and non-insulin dependent diabetes.
  • PCI/3 antigen and “PC2 antigen” as used herein are inclusive terms that mean not only the PCI/3 or PC2 enzymes themselves but any peptide segment thereof reactive with the disclosed antibody, such as the amino acid sequences 114-158 of PCI/3 and 112-159 of PC2.
  • labeled is used herein as it is ordinarily understood in the art, to include attachment or a binding of an atom or molecule which provides a detectable signal.
  • labels include, but are not limited to, a radioisotope, a fluorescent molecule (fluorophore) , or dye molecule (chromophore) , chemiluminescent molecule, an enzyme (which can catalyze a reaction that produces a detectable product or consumes a detectable substrate) , or a ligand whose specific binding provides a detectable or measurable result.
  • immunological methods of detection or measurement include formation of an antigen-antibody complex which is separated from unbound antigen or unbound antibody, depending upon which is in excess in the reaction.
  • Many separation techniques are known to those skilled in the art, the choice of which depends upon the specific assay employed.
  • separation techniques include precipitation, electrophoresis, or binding to a solid matrix.
  • solid matrix include beads, cell membranes, reaction vessel walls, gels, filter surfaces and the like.
  • Detection or measurement is carried out by means appropriate to the nature of the label providing the detectable signal .
  • Quantitative measurement entails providing control samples having predetermined amounts of antigen such that a standard curve is obtained.
  • the presence or amount of antigen- antibody complex or unbound antigen is then correlatable to the control samples having predetermined presence or amount of antigen.
  • antigen- antibody complex or unbound antigen There is at present a large variety of techniques and expedients available to those skilled in the art, as set forth in various books and publications of immunological methods, for example "Antibodies, a Laboratory Manual” by E. Harlow and D. Lane, Cold Spring Harbor Laboratory (1988) .
  • the antibodies of the present invention can be either monoclonal or polyclonal.
  • Fig. 1 is a standard curve for the PCI/3 RIA comprising pg x IO "4 of PCI/3 (vertical axis) and radioactivity counts (log scale, horizontal axis) .
  • Fig. 2 is a standard curve for the PC2 RIA comprising pg x
  • the human PCI/3 primers were designed to amplify the nucleotide region 547-681 encoding amino acids 114-158 (SEQ ID NO:5)
  • the human PC2 primers were designed to amplify the region 421-564 encoding amino acids 112-159 (SEQ ID NO:6)
  • Both sets of primers contained 5' BamHI and 3 'EcoRI sites for later use in ligation into the multiple cloning site contained in the prokaryotic expression vector pGEX-2T (Pharmacia Inc., Piscataway, NJ) .
  • RT-PCR was used to amplify both PCs from mRNA isolated from the human medullary thyroid carcinoma cell line (hMTC; obtained from Dr. H.
  • the PCR products were purified and ligated into the vector.
  • the plasmids which contained the desired constructs were then expressed in bacteria.
  • the resulting fusion protein products were purified by glutathione affinity chromatography and sent to HRP Inc. (Denver, PA) for polyclonal antibody production.
  • the fusion protein is comprised of glutathione-S-transferase (GST) (Sigma Chemicals, St. Louis, MO) linked to a thrombin cleavage site which is then attached to the antigen of interest, in this case the N-terminal catalytic portion of either PCI/3 or PC2.
  • the GST-directed antibodies were separated from whole sera using affinity columns containing GST bound to Affigel-10 resin (BioRad, Hercules, CA) .
  • the GST affinity columns consisted of two 4 ml layers; layer A contained unpurified GST (GST protein and other assorted bacterial proteins) and layer B consisted of purified GST.
  • the initial titer and specificity of the sera were determined by Western blot analysis using Chemiluminescence Renaissance kit (DuPont NEN, Wilmington, DE) and by RIA.
  • the RIAs for both PCI/3 and PC2 were developed using synthetic peptide fragments of the original antigen used for immunization which were made at the Emory University Microchemical Facility.
  • the peptides synthesized corresponded to residues 114 to 136 for PCI/3 (designated PC1/3-N23) and 112 to 136 for PC2 (designated PC2-N25) .
  • Norleucine was substituted for methionine in each peptide.
  • the PCI/3 and PC2 fragments were iodinated using a chloramine-T procedure (Milgram, S.L., et al. (1989) Peptides 10:1013-1017) except that 1 mCi [ I25 I]Nal (>400 mCi/ml, ICN, Costa Mesa, CA) was used and the reaction was terminated after one minute.
  • the labeled material was then run over an Econo-Pac 10DG desalting column (Bio-Rad) and aliquots from 200 ⁇ l fractions were counted on a gamma counter.
  • the void volume peak tube plus one fraction on either side were pooled, diluted in assay diluent (AD) and stored at 4°C.
  • the AD contained 9 mM EDTA (Fisher, Atlanta, GA) , 0.3% Fraction V BSA (Sigma) , and 0.01% Na-Azide (Sigma) in 0.05 M phosphate buffer, pH 7.4.
  • the total volume of each assay is 400 ⁇ l, with 200 ⁇ l of standard or unknown, 100 ⁇ l of antibody, and 100 ⁇ l of labeled peptide standard (10,000 cpm/100 ⁇ l) , all diluted in AD and added on the same day.
  • Final antibody dilutions were 1:8,000 for PCI/3 and 1:12,000 for PC2, with each giving approximately 25% binding in the absence of unlabeled ligand.
  • the standard concentrations ranged from 10 to 1,280 pg for PCI/3 and from 5 to 1,500 pg for PC2.
  • the unbound antigen was precipitated by adding 200 ⁇ l AD containing 0.25% dextran (Sigma) and 1% charcoal (Mallinckrodt, Paris, KY) , incubating for 20 minutes at 4°C, then centrifugation at 3,600 rpm at 4°C for 30 minutes in a Sorvall RT6000 centrifuge. The supernatant is carefully aspirated and the pellets counted for one minute in the gamma counter, using LKB-Wallac software to analyze the standard curve by log-logit transformation. Results are shown for PCI/3 in Fig.
  • PCI/3 antisera Both affinity-purified and unpurified PCI/3 (EM4 and EM5) and PC2 (EM6 and EM7) antisera were examined by Western analysis and RIA to determine specificity. Both PCI/3 antisera showed cross-reactivity by Western analysis with the construct GST- PC1/3, but not with GST-PC2, while both PC2 antisera recognized the GST-PC2 construct, but not GST-PC1/3 (data not shown) . RIAs were performed to further assess the specificity of the PCI/3 and PC2 antibodies. Cross-reactivities of the PC constructs and various synthetic peptides in the PCI/3 and PC2 constructs and various synthetic peptides in the PCI/3 and PC2 RIAs were determined.
  • the PCI/3 RIA having a sensitivity of 10 pg, exhibited 0.0002% cross-reactivity with PC2-N25 and 0.0001% with GST-PC2 at concentrations up to 10 ⁇ g.
  • the PC2 RIA having a sensitivity of 5 pg, showed 0.08 and 0.02% cross-reactivity with PC1-N23 and GST-PCl/3, respectively, at concentrations up to 10 ⁇ g.
  • Neither the PCl/3 nor PC2 RIA exhibited cross-reactivity with GST. All other synthetic peptides (16 different islet, brain and gut derivatives) examined in the PCI/3 and PC2 RIAs exhibited ⁇ 0.003 and 0.007% cross-reactivity, respectively (see Table 1) .
  • the antibodies disclosed herein are directed to a region of each PC that includes the N-terminus of the catalytic domain, specifically the region encoded by nucleotides 547-681 of the hPCl/3 coding region, and 421-564 of the hPC2 coding region.
  • the unique sensitivity and specificity of the described antibodies is considered to be due, at least in part, to the choice of the region to which antibodies were raised. Therefore it will be understood by those skilled in the art that other antibody preparations directed at least in part against peptide segments contained within the described regions will have equivalent properties to the antibodies described herein and are included within the present invention.
  • Such equivalent antibodies can be directed against peptide segments lying wholly within or overlapping the peptide segments described herein.
  • monoclonal antibodies directed against epitopes within the described peptide regions can be selected, having equivalent or improved properties of sensitivity and/or specificity.
  • the antibodies described herein can be made by a variety of techniques besides the one specifically described.
  • the animal species to be immunized to produce the antibody can be varied as desired, depending on convenience, amount of antibody required, and other characteristics as known in the art.
  • Other means for obtaining the described immunogenic peptide region for each PC can be employed as convenience and efficiency dictate.
  • peptide and antibody purification methods can be varied, as deemed appropriate, to achieve desired levels of purity.
  • the antibody can be used for a large variety of applications.
  • the antibodies described herein can be used in other forms of radioimmunoassay, enzyme-linked immunoassays (ELISA) , fluorescence-tagged applications including immunostaining, "Western” gel electrophoresis, affinity separation and the like.
  • ELISA enzyme-linked immunoassays
  • fluorescence-tagged applications including immunostaining, "Western” gel electrophoresis, affinity separation and the like.
  • the described antibodies can be used in any of the vast array of immunochemical methods known in the art, as described by standard texts such as Harlow, E. and Lane, D. Antibodies, a Laboratory Manual . Cold Spring Harbor Laboratory (1988) .
  • the antibodies described herein have been used to demonstrate the existence of PCI/3 and PC2 in normal human serum, a result previously unobserved with prior antibodies.
  • a normal range of 220.7 ⁇ 82.9pg/ml of PCI/3 and 103.7 ⁇ 27.7pg/ml of PC2 have been observed.
  • Assays can be carried out in 200 ⁇ l of serum. Such levels are within the detectable range of the described antibodies. Sensitivity can be f rther improved by sample pretreatment, for example by concentrating the sample; however, best results for serum assays have been achieved with non-concentrated serum.
  • Such diseases can include familial hyperproinsulinemia, hyperproglucagonemia, non-insulin dependent
  • Type II diabetes increased prohormone expression observed in pancreatic and other cancers, and polyendocrine disease characterized by defective hormone processing. See, e.g., O'Rahilly, S. et al. (1995) Abstr. 77th Ann. Mtg. Endocrine Soc. 597.- Yoshida, H. et al. (1995) Diajbetes 4 :389-393; Kahn, S.E. et al. (1995) Diabetes ____ :173-179; Pour, P.M. (1995) Int . J. Pancreatology 12:217-223; Bertagna, X. (1994) Endocrinol . and Metabolism Clinics of N. America 23.467-485.
  • Kits for carrying out an immunoassay for detecting and measuring PCl/3 or PC2 can be provided using components described herein, packaged in unit assay form for user convenience.
  • Such kits contain, at a minimum, antibody to the region of PCI/3 encoded by nucleotides 547-681 or to the region of PC2 encoded by nucleotides 421-564 or equivalent regions thereof, in an amount sufficient to detect at least 10 pg PCI/3 or 5 pg PC2 in a 200 ⁇ l sample.
  • kits can include pre-packaged dilution buffer, standard dilutions of known concentrations of PCI/3 or PC2 or the peptides segments thereof specific to the respective antibodies, as described herein (PC1/3-N23 or PC2- N25) .
  • :2S I-labelled PC1/3-N23 or PC2-N25 or equivalents thereof can be provided.
  • Each component can be separately packaged in unitary form to maximize ease of use and minimize measurement and transfer.
  • Materials for adsorbing or precipitating unbound antigen can be included, also prepackaged in an amount appropriate for each assay.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Urology & Nephrology (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des anticorps agissant spécifiquement contre la prohormone convertase 1/3 (PC1/3) et la prohormone convertase 2(PC2). Les anticorps permettent de descendre la sensibilité du dosage radioimmunologique à 10 pg de PC1/3 ou à 5 pg de PC2 dans l'échantillon. Ces dosages ont permis de déterminer quels étaient les niveaux sériques normaux de PC1/3 et de PC2. L'invention concerne également un procédé permettant de diagnostiquer une maladie liée à des taux anormaux de PC.
PCT/US1996/014800 1995-09-15 1996-09-13 Dosages radioimmunologiques par anticorps des propeptide/prohormone convertases pc1/3 et pc2 WO1997010504A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU70724/96A AU7072496A (en) 1995-09-15 1996-09-13 Antibody radioimmunoassays for human propeptide/prohormone convertases pc1/3 and pc2

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US378295P 1995-09-15 1995-09-15
US60/003,782 1995-09-15

Publications (1)

Publication Number Publication Date
WO1997010504A1 true WO1997010504A1 (fr) 1997-03-20

Family

ID=21707578

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/014800 WO1997010504A1 (fr) 1995-09-15 1996-09-13 Dosages radioimmunologiques par anticorps des propeptide/prohormone convertases pc1/3 et pc2

Country Status (2)

Country Link
AU (1) AU7072496A (fr)
WO (1) WO1997010504A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006010531A1 (fr) * 2004-07-28 2006-02-02 F. Hoffmann-La Roche Ag Prohormone convertase 1 utilisee en tant que marqueur/cible pour la defaillance des cellules beta

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FEDERATION OF EUROPEAN BIOCHEMICAL SOCIETIES LETTERS, March 1994, Volume 344, BLACHE et al., "Immunological Detection of Prohormone Convertases in Two Different Proglucagon Processing Cell Lines", pages 65-68. *
JOURNAL OF BIOLOGICAL CHEMISTRY, 15 March 1993, Volume 268, No. 8, ZHOU et al., "Purification and Characterization of the Prohormone Convertase PC1(PC3)", pages 5615-5623. *
JOURNAL OF BIOLOGICAL DISEASES, February 1995, Volume 270, ROTHENBERG et al., "Processing of Mouse Proglucagon by Recombinant Prohormone Convertase 1 and Immunopurified Prohormone Convertase 2 In Vitro", pages 10136-10145. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006010531A1 (fr) * 2004-07-28 2006-02-02 F. Hoffmann-La Roche Ag Prohormone convertase 1 utilisee en tant que marqueur/cible pour la defaillance des cellules beta

Also Published As

Publication number Publication date
AU7072496A (en) 1997-04-01

Similar Documents

Publication Publication Date Title
Melani et al. Human serum proinsulin
Oyer et al. Studies on human proinsulin: Isolation and amino acid sequence of the human pancreatic C-peptide
Gametchu et al. Use of receptor antibodies to demonstrate membrane glucocorticoid receptor in cells from human leukemic patients
Pavlakis et al. Expression of two human growth hormone genes in monkey cells infected by simian virus 40 recombinants.
US7264938B2 (en) Method of detecting native proBNP
US6929924B2 (en) C-peptide specific assay procedure
Rothenberg et al. Processing of Mouse Proglucagon by Recombinant Prohormone Convertase 1 and Immunopurified Prohormone Convertase 2 in Vitro (∗)
US20050214883A1 (en) Novel alanine transaminase enzyme and methods of use
Ratcliffe et al. Development and validation of an immunoradiometric assay of parathyrin-related protein in unextracted plasma
WO1992007068A1 (fr) Protease d'amylïodine et ses emplois
EP1456234A2 (fr) Regions des epitopes du recepteur de la thyrotrophine (tsh) leurs utilisations et leurs anticorps
WO1989009222A1 (fr) Topoisomerase i humaine clonee
WO1990008195A1 (fr) Anticorps monoclonal se liant au collagene humain de type ix
CA2257858A1 (fr) Procede pour le diagnostic et le pronostic du cancer
US6770460B1 (en) Fusion protein and its use in an immunoassay for the simultaneous detection of autoantibodies related to insulin-dependent diabetes mellitus
US5773237A (en) Method for determining tyrosine kinase activity
WO1998054218A1 (fr) Formes de troponine i et leur utilisation
US5663315A (en) Isolated DNA encoding human GP2
EP1021721B1 (fr) Dosages des anticorps du recepteur de la tsh
WO1995007464A1 (fr) Diagnostic du diabete insulinodependant
WO1996017873A9 (fr) Diagnostic de la pancreatite
WO1997010504A1 (fr) Dosages radioimmunologiques par anticorps des propeptide/prohormone convertases pc1/3 et pc2
US5942402A (en) Method of detecting and treating cancer
EP0393640A1 (fr) Anticorps monoclonaux reconnaissant l'extrémité C-terminale de l'ANP
WO1999043710A1 (fr) Antigenes d'enveloppe specifiques a la prostate et procedes de preparation et d'utilisation desdits antigenes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i

Free format text: PAT.BUL.13/97 UNDER "PUBLISHED", ADD "WITH A REQUEST FOR RECTIFICATION UNDER RULE 91.1(F)"

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
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA